Jun 22, 2026

Comprehensive protocols for H1-10 nucleolar functional characterization

DOI
https://dx.doi.org/10.17504/protocols.io.3byl4m73olo5/v1
  • Xiaoyu Liu1,2,3,
  • Wenzheng Wang4,
  • Xianglin Zhang1,2,3,
  • Haiyang Guo1,2,3
  • 1Department of Clinical Laboratory, the Second Hospital, Cheeloo College of Medicine, Shandong University, Jinan, Shandong, China;
  • 2State Key Laboratory of Discovery and Utilization of Functional Components in Traditional Chinese Medicine, Shandong Engineering & Technology Research Center for Tumor Marker Detection, the Second Hospital, Cheeloo College of Medicine, Shandong University, Jinan, Shandong, China;
  • 3Shandong Provincial Clinical Medicine Research Center for Clinical Laboratory, the Second Hospital, Cheeloo College of Medicine, Shandong University, Jinan, Shandong, China;
  • 4Institute of Medical Sciences, the Second Hospital, Cheeloo College of Medicine, Shandong University, Jinan, Shandong, China
Xiaoyu Liu
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DOI: https://dx.doi.org/10.17504/protocols.io.3byl4m73olo5/v1
Protocol Citation: Xiaoyu Liu, Wenzheng Wang, Xianglin Zhang, Haiyang Guo 2026. Comprehensive protocols for H1-10 nucleolar functional characterization. protocols.io https://dx.doi.org/10.17504/protocols.io.3byl4m73olo5/v1
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License: This is an open access  protocol  distributed under the terms of the  Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: June 18, 2026
Last Modified: June 22, 2026
Protocol  Integer ID: 319389
Keywords: nucleolar functional characterization, nucleolar distribution measurement, cell functional assay, comprehensive protocols for h1, tissue microarray analysis, bioinformatic analytical pipeline, functional assay, chemoproteomic, h1, protein purification
Disclaimer
This overview protocol is provided for research use only. Individual experimental procedures may require optimization depending on experimental conditions.
Abstract
This unified protocol documents a set of wet-lab experiments and bioinformatic analytical pipelines. Independent sections cover cell functional assays, lentiviral and fusion plasmid construction, in vitro protein purification and phase separation, immunoprecipitation and chemoproteomics, nucleolar distribution measurement, compound screening, tissue microarray analysis, and ChIP-Seq/RNA-Seq data processing.
Western blotting
Cells were harvested and lysed on ice using a RIPA buffer (Beyotime, P0013B) containing 1% PMSF (Solarbio, P0100). The proteins were purified by centrifugation (12,000 g at 4 °C for 20 min) and quantified by Bicinchoninic Acid (BCA) protein assay. Total protein concentrations were equaled in all samples. The proteins were heated at 95 °C for 10 min in the loading buffer (Beyotime, P0015L), then loaded onto a 10% sodium dodecyl sulfate (SDS)-polyacrylamide electrophoresis gel, electrophoresed with Tris-glycine running buffer at 15 V/cm for 1 h, and finally transferred to a polyvinylidene difluoride membrane (Millipore, IPVH00010). This membrane was incubated at room temperature (RT) in a blocking buffer (5% non-fat dry milk in TBST) for 2 h. After incubated with primary antibodies, the membranes were washed three times with TBST and incubated with corresponding secondary antibody for 1 h at RT.
Cell proliferation assay
Agilent xCELLigence RTCA (Real-Time Cell Analysis) was employed for real-time monitoring of cell growth rate. Briefly, 8000 cells per well were cultivated into the E-plate and placed in the RTCA station for continuous monitoring over a period of 96 h.
Plate colony formation assay and soft agar colony formation assay
For plate colony formation, cells were seeded in 6-well plates and incubated for 14 days in complete culture media. Subsequently, the cells were fixed with methanol and stained with 0.1 % crystal violet solution. For soft agar colony formation, 2 ml medium containing 0.6 % agar was used as the bottom layer. Cells were suspended in 2 ml medium containing 0.3 % agar and then poured onto the bottom layer. After 3 weeks, the colonies were stained with 0.1% crystal violet solution. The number of clones was analyzed using ImageJ software.
Lenti-viral vector construction and transfection
To generate the lentiviral vector pLVX-IRES-Puro-H1-10-Flag, the coding sequence (CDS) of H1-10 was amplified, with the addition of a 1x flag sequence in the reverse amplification primer, and subsequently inserted into pLVX-IRES-Puro vector. To generate the lentiviral vector pLKO.1-TRC-shH1-10, two short hairpin RNA (shRNA) oligonucleotides targeting distinct regions of H1-10 were individually inserted into the pLKO.1-TRC vector (Addgene, 10878). All constructs were sequenced to ensure sequence identity. The lentiviral vectors above were respectively co-transfected with psPAX2 and pMD2G vectors into HEK293T cells using Lipofectamine 2000 transfection reagent. Supernatants were collected at 48 h and 72 h post-transfection. After concentration using PEG8000, viral particles were resuspended, aliquoted and stored at -80°C. For infection, cells were seeded at an optimal density one day prior, followed by the addition of the viral to the culture medium on the subsequent day.
To generate H1-10 overexpression vector pEGFP-C2-H1-10, the CDS of H1-10 was cloned into pEGFP-C2 vector. The CDS of the remaining genes in the H1 variants was inserted using a similar approach.
To construct recombinant plasmid for H1-10 expression in Escherichia coli, H1-10 coding region was subjected to codon optimization to facilitate expression in E. coli, and a 14-amino acid linker sequence "GAPGSAGSAAGGSG" was appended to the N-terminus of H1-10. The above bases were synthesized by BioSune biotechnology and then inserted into the pET28a-EGFP using In-Fusion cloning using ClonExpress II One Step Cloning Kit (Vazyme, C11201). The successfully constructed pET28a-EGFP-H1-10 vector includes a 5’ 6xHIS followed by mEGFP, a 14 amino acid linker sequence and H1-10 coding region. Similar approaches were employed for the construction of pET28a-mEGFP-FBL and pET28a-mCherry-NPM1. For the truncations of pET28a-EGFP-H1-10, they were generated respectively using the fast mutagenesis system (TransGen, AS221-01). When designing the extension portion of the forward primer, the targeted deletion region should be skipped.
mCherry-H1-10 and iLOV-H1-10 fusion protein vector construction
The pET28a-iLOV and pET28a-iLOV-H1-10 plasmids were synthesized by Tsingke biotechnology, with the CDS regions of the proteins inserted into the pET28a plasmid respectively. pET28a-mCherry was purchased from Miaoling Biology and the CDS region of H1-10 was cloned into it to generate the plasmid pET28a-mCherry-H1-10.
In vitro protein expression, purification, phase-separation, and nucleolar assembly experiments
For protein expression, all expression constructs were transformed into BL21 cells individually. A fresh bacterial colony was inoculated into LB media supplemented with kanamycin and cultivated overnight at 37 °C. The cells were diluted in a ratio of 1:30 in 500 ml LB containing kanamycin, and grown for 3 hours at 37 °C until OD600 reached the range of 0.6 to 0.8. Following this, 1 mM IPTG was added into the cells, and the cells were further cultured for 18 hours at 16 °C. Afterward, the cells were harvested and stored at -80 °C. For protein purification, cells were resuspended in 20 ml cold Lysis Buffer (50 mmol/L Tris-HCl, pH=7.5; 500 mM NaCl; 5mM Imidazole) containing 1 mM PMSF and followed by homogenization using sonication in an ice bath (3 s on, 5 s off, Amp%=33%) for 40 min. The lysates were cleared by centrifugation at 12,000 x g for 30 min 4 °C and added to the 1 ml pre-equilibrated Ni-NTA agarose with 5 volumes of Lysis Buffer. And the effluent was collected and subjected to a repeated loading. Then the slurry was washed with 15 volumes of Wash Buffer (50 mmol/L Tris-HCl, pH=7.5; 500 mM NaCl; 20mM Imidazole) until the non-specific proteins were completely eluted. 5 volumes of Elution Buffer (50 mmol/L Tris-HCl, pH=7.5; 500 mM NaCl; 200mM Imidazole) was added and the effluent was collected. Amicon Ultra centrifugal filters (Millipore) were employed to concentrate the protein and exchange the buffer to high salt (HS) buffer (25 mmol/L HEPES, pH 7.5; 500 mmol/L NaCl; 1 mmol/L DTT).
To induce phase separation, a low salt (LS) buffer (25 mmol/L HEPES, pH 7.5; 1 mmol/L DTT) was prepared. Recombinant proteins were then added to specified amounts of both HS buffer and LS buffer, which contained a molecular-crowding agent, PEG-8000. This resulted in the proteins being present in the Droplet Formation Buffer (25 mmol/L HEPES, pH 7.5; 150 mmol/L NaCl; 1 mmol/L DTT; 10% PEG-8000). Once induced, 10 µl of protein solution was taken onto a glass slide, and the formation of the liquid phase was immediately observed under a confocal microscope (63 x oil immersion; Zeiss LSM 800, Zeiss, Germany). For fluorescence recovery after photobleaching (FRAP) assay, 10 µl of protein solution was added onto a cell culture dish, followed by laser-induced bleaching of the region of interest (ROI). Subsequently, the recovery time of fluorescence in the ROI was measured.
Co-immunoprecipitation and mass spectrometry assay
This experiment was performed as previously described1. Shortly, cells were washed with PBS and lysed in lysis buffer containing protease inhibitors (50 mM Tris-HCl pH8.0, 150 mM NaCl, 2 mM EDTA, 0.3% IGEPAL CA-630) at 4 °C for 30 min. Protein A/G beads were washed and incubated with appropriate antibodies at RT for 30 min. The cell lysate was added to the antibody conjugated beads and incubated at 4 °C overnight. The antigen-antibody complex was washed 10 times with TBST (0.1% Tween 20 in TBS) and eluted using 50 µL 1×SDS-PAGE loading Buffer at 95 °C for 5 min. For sample preparation before mass spectrometry, the target bands were carefully excised into approximately 1mm³ fragments using a scalpel. Decolorization was performed by adding 100 mM NH4HCO3/30%ACN at RT. Once the gel became colorless, the supernatant was discarded, and the gel was frozen dry. Each tube was supplemented with 90 mM NH4HCO3 and 10 mM DTT, followed by incubation at 56 °C for 30 min to facilitate protein reduction. The supernatant was discarded, and 100% ACN was added. After discarding the supernatant once again, 70 mM NH4HCO3 and 60 mM freshly prepared IAA were added and kept in the dark for 20 min at RT. After discarding the supernatant, 100 mM NH4HCO3 was employed to each tube for 15 min at RT. Following another supernatant removal, the gel was treated with 100% ACN for 5 minutes RT and subsequently freeze-dried. After drying, 5 µl 2.5-10 ng/µl trypsin solution was added and incubated at 4 °C for 30-60 min. Then 50 mM NH4HCO3 was added and the mixture was incubated at 37 °C overnight. The following day, the digested liquid was transferred to a new tube, while the original tube was treated with 60% ACN/0.1% TFA and sonicated for 15 min. The solution was pipetted out and incorporated into the previous solution. And the extraction was repeated 3 times. Finally, all solutions were combined and subjected to freeze-drying. With this, the sample preparation process was completed, and the sample could be redissolved and used for mass spectrum analysis.
GST-pull downGST-pull down
The optimized coding sequence of H1-10 was cloned into the pGEX4T-1 expression vector. The correctly sequenced plasmid above and the control plasmid were transformed into BL21 cells. The way to express GST fusion proteins was analogous to the aforementioned protein expression approach. Cells were collected, resuspended and sonicated. Then lysates were cleared by centrifugation and then added to pre-cleaned glutathione beads (Beaver Biotechnology, 70601-5). After incubation for 1 h to make the GST-fusion proteins fully arrested by beads, 22Rv1 cell lysate was then incubated with glutathione beads for another 4 h at 4 °C. Later, beads were rinsed by cold PBS for 10 times and boiled in SDS loading buffer. The bound proteins were loaded onto a 10% SDS-PAGE, followed by analysis of Western blot using an anti-Pol I or anti-UBF1 antibody.
Cell cycle synchronization and quantification of H1-10 nucleolar/nucleoplasm distribution
22Rv1 cells were treated with 5 mM sodium butyrate (MCE, HY-B0350A) for 24 h to arrest the cell cycle in G1 phase. To synchronize the cell cycle in S phase, they were incubated with 10 µM aphidicolin (MCE, HY-N6733) for 24 h and subsequently released from this block by washing with PBS. The cells were then grown in regular medium for different durations. Early S-phase cells were harvested 2 h after release from the aphidicolin block, while mid-S-phase cells were harvested at 6 h. To arrest them in G2/M phase, they were exposed to 100 ng/ml nocodazole for 16 h. Cells were immunostained with rabbit anti-H1-10 and mouse anti-NPM1 primary antibodies and photographed with a confocal microscope. To quantify the distribution of H1-10 between the nucleolus and nucleoplasm, using the profile tool in Zeiss software, lines were drawn and the location of nucleoli was indicated by NPM1 staining. Subsequently, fluorescence intensities of H1-10 within the nucleolus and nucleoplasm along these lines were obtained.
Polysome Profiling Analysis
Polysome profiling analysis could be roughly divided into four parts, namely gradient preparation, cell lysate preparation, ultracentrifugation and fraction collection, RNA isolation and RT-PCR. For gradient preparation, 10% sucrose and 50% sucrose solution each in 1 x sucrose gradient buffer (20 mM HEPES, pH 7.6;100 mM KCl; 5 mM MgCl2; 10 µg/ml cycloheximide; 1 x protease inhibitor cocktail;10 units/ml RNase inhibitor) were respectively prepared. Gradient Station (Biocomp) was utilized for the preparation of a homogeneous linear gradient of sucrose ranging from 10% to 50% and the tubes (Beckman Coulter, 344059) should be carefully taken out and stored vertically at 4 °C for 2 h without disturbance. For cell lysate preparation, 80-90% confluent 22Rv1 cells with or without H1-10 stable overexpression in a 15 cm dish were prepared and treated with 100 µg/ml cycloheximide for 5 min. Cells were washed twice with ice-cold PBS and harvested by scraping into PBS containing 100 ug/ml CHX. Cells were then collected by centrifuging and resuspended in 500 µl of hypotonic buffer (5 mM Tris-HCl, pH 7.5; 2.5 mM MgCl2; 1.5 mM KCl; 100 µg/ml CHX; 2 mM DTT; 0.5% Triton X-100; 0.5% Sodium Deoxycholate; 100 units of RNasin inhibitor; 1x protease inhibitor cocktail). After centrifuging at 4 °C, the supernatant was transferred to a pre-chilled 1.5 ml tube and OD260nm for each sample was measured using NanoDrop. For the third part, ultracentrifugation and fraction collection, 30-50 OD260 equivalent of lysate was gently loaded onto each gradient and samples were ultracentrifuged at 180,000 x g for 2 h at 4 °C (Beckman Coulter, SW40Ti rotor). And the fractionation in 1.5 ml tubes was runed following the manufacturer’s instructions of Biocomp. For the last part, 1 ml Trizol was added to each 1.5 ml tube, and RNA was isolated in accordance with the manufacturer’s instructions. Equal volumes of RNA were subjected to cDNA synthesis, and equivalent cDNA was used as a template for PCR.
Nascent RNA synthesis
In vivo metabolic pulse labeling was a classical approach to assess production and processing of nascent RNA. Here 4-thiouridine (4sU, Sigma-Aldrich, T4509) labeling was used to separate newly synthesized RNA from the preexisting RNA. On the previous day, cells were seeded on a 10 cm cell culture dish one day in advance and cultured to a 60-80% confluence. Cells were treated with 100 µM final concentration of 4sU for 2 h to achieve metabolic pulse labeling of nascent RNA. Then cold TRIzol reagent (Invitrogen, 15596026) was added into dishes and total RNA isolation was based on standard TRIzol RNA extraction protocol. 1 mg/ml biotin-HPDP (Thermo Scientific, A35390) in DMF was specifically prepared for 4sU-labeled total RNA biotinylation reaction. After incubation at RT for 3 h in dark, the biotinylated RNA was purified using Phase-Lock-Gel tubes (TIANGEN, WM5-2302830). At this stage, biotinylated, 4sU-tagged RNA (nascent RNA) and nontagged RNA (steady-state) were both contained in the RNA sample. And the separation of 4sU-tagged RNA from nontagged RNA was conducted as the manufacturer’s instructions of µMacs Streptavidin Kit (Miltenyi, 130133282). Briefly, RNA samples were denatured at 65 °C for 10 min and on ice immediately for 5 min, followed by incubation with µMacs Streptavidin MicroBeads at RT for 15 min. Subsequently, samples were loaded to pre-equilibrated µMACS column and the flow through contained nontagged RNA. Freshly prepared 100 mM DTT was used to elute the biotin-4sU labeled RNA from the column. At last, ethanol precipitation was employed to recover the preexisting RNA and newly transcribed RNA, followed by analysis with agarose gel electrophoresis.
Nucleolar DNA sequencing and NAD identification
Cells were cross-linked by 1% formaldehyde and quenched with 125 mM glycine upon reaching 80% confluence. Then they were resuspended in 2 ml of buffer A (10 mM HEPES, pH 7.9; 10 mM KCl; 1.5 mM MgCl2; 0.5 mM DTT) and incubated on ice for 5 min. The cell suspension was transferred to a precooled 2-ml Dounce tissue homogenizer and homogenized 10 times. Subsequently, the homogenized cells were centrifuged and rough nuclei were contained in the pellets. The pellets were resuspended with 3 ml S1 solution (0.25 M sucrose; 10 mM MgCl2), and the liquid was then carefully layered over 3 ml S2 solution (0.35 M sucrose; 0.5 mM MgCl2) ensuring that the two layers remained distinctly separated. The mixture was centrifuged at 2500 x g for 5 min at 4 °C to obtain a cleaner nuclear pellet. The pellets were resuspended with 3 ml S2 solution and subjected to sonication with six 10-s bursts to disrupt nuclear membranes. The liquid was then layered over 3 ml S3 solution (0.88 M sucrose; 0.5 mM MgCl2), and then centrifuged at 3500 x g for 10 min at 4 °C. The supernatant was discarded, and the pure isolated nucleoli were collected. Total genomic and nucleolar DNA was isolated with TIANamp Genomic DNA Kit (TIANGEN, DP304) and dissolved in TE buffer (10 mM Tris, 1 mM EDTA, pH 7.9). After shearing the DNA by sonication, libraries were constructed using ThruPLEX DNA-seq kit (Rubicon Genomics).
Raw sequencing data of nucleolar and genomic DNA were mapped to the hg19 reference genome using Bowtie2, and the output bam files were converted to bigwig files using the BamCoverage function of deepTools with 1 x normalization (RPGC). Based on this, nucleolar enrichment was determined by calculating the log2 ratio of the nucleolar signal to the genomic background signal. Subsequently, the bigWigToBedGraph function was to convert the log2-transformed bigwig file into bedgraph format. Next, the average nucleolar enrichment in each 50 kb bin within the bedgraph file was calculated and defined as the “NAD scores”. These scores were used to identify NADs (NAD scores > 0.5) and non-NAD regions across the genome.
Tissue microarray
The prostate cancer tissue microarray (HProA150CS01) was purchased from Shanghai Outdo Biotech Company. This research received ethical approval from the Ethics Committee of the company (Approval No: YB M-05-01). Immunohistochemical staining was conducted to assess the expression of H1-10 in prostate cancer and adjacent non-cancerous tissues. Firstly, the microarray was deparaffinized using xylene and gradient alcohol, followed by antigen retrieval with EDTA buffer and blocking of endogenous peroxidase activity with hydrogen peroxide solution. Following incubation with H1-10 antibody (Abcam, ab31972) and secondary antibody, DAB staining was performed, followed by counterstaining with hematoxylin and mounting. The tissue microarray was subsequently captured using a Nanozoomer Digital Pathology scanner (Hamamatsu, NanoZoomer S60). To analyze the tissue microarray cores, the TMA plugin in Halo v3.0.311.314 analysis software was employed to determine the diameter and row-column numbers. The Indica Labs - Area Quantification v2.1.3 module was utilized to quantify various parameters within the target region of each slide, including tissue area (μm²), weak positive area (μm²), moderate positive area (μm²), and strong positive area (μm²). The H-Score was calculated as follows: H-SCORE=∑(PI×I) = (percentage of cells with weak intensity × 1) + (percentage of cells with moderate intensity × 2) +(percentage of cells with strong intensity × 3), where pi represents the proportion of positive signal area and i represents staining intensity. All patients provided written informed consent for the use of their tissue samples.
Virtual screening for H1-10 antagonists and In vitro compounds treatment
The structure-based virtual screening was performed using Schrodinger Maestro 11.4. The proteins and ligands were prepared before run docking. The 3D structures of human linker histone H1-10 (PDB ID: 7K602), H1-4 (PDB ID: 7K5Y2) and H1-0 (PDB ID: 7K5X2) were download from the Protein Data Bank (https://www.rcsb.org/). The DNA-binding domain near the N-terminal region of H1-10, H1-4 and H1-0 was located at the α2 helix, and consisted of DNA-binding residues of Ala 67, Ala 59 and Gln47, respectively. After retrieving protein structure, the protein preparation wizard panel was executed with the OPLS_2005 force field. The receptor energy minimization was terminated when the energy con-verged or the root mean square deviation (RMSD) reached a maximum cutoff 0.30 Å. The prepared protein structure was further subjected to gird generation. The DNA-binding residue were selected as the centroid of the grid box (20 Å ✕ 20 Å ✕ 20 Å) in receptor grid generation panel. About 1.6 million compounds were incorporated for ligand preparation into the LigPrep panel by using following criteria (i) select the OPLS-2005 force field, (ii) generate possible states with ionizer at target pH 7.0 ± 2.0, (iii) choose the desalt option, (iv) generate tautomers, (v) generate at most 32 conformations and retain specified chiralities (vary other chiral centers). The prepared ligands were subjected to virtual screening including HTVS with 90% cutoff, SP with 90% cutoff and XP with 90% cutoff. Subsequently, HTVS programs using the H1-10 binding compounds as ligand, H1-0 and H1-4 as receptor were executed. The H1-0 and H1-4 binding ligands were excluded to eliminate nonspecific binding. Finally, all H1-10 specific binding ligands were sorted by docking score, and the top 50 ligands with higher docking score were selected for the further research. Compounds property of pan-assay interference compounds (PAINS) was analyzed using SwissADME. Forty-six available compounds from top 50 HTVS hits and two negative control compounds were purchased from TargetMol (USA) and dissolved in DMSO to 100 mM as the stock solution. The 22Rv1 cells were seeded overnight and treated with 0120-0018 (78 μM), D271-0003 (92 μM) or DMSO for 48h for RNA purification, SUnSET and dual-luciferase assay.
Synthesis of biotin-CN27
In brief, 5-fluoro-2-nitroaniline (1) and tert-butyl (3-bromopropyl)carbamate (2) underwent substitution reaction to obtain tert-butyl (3-((5-fluoro-2-nitrophenyl)amino)propyl)carbamate (3), which was reduced by hydrogen to obtain tert-butyl (3-((2-amino-5-fluorophenyl)amino)propyl)carbamate (4). Compound 4 underwent condensation with 4-(((7-methyl-7H-pyrrolo[2,3-d]pyrimidin-4yl)amino)methyl)benzoic acid (5) to afford tert-butyl (3-((5-fluoro-2-(4-(((7-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)methyl)benzamido)phenyl)amino)propyl)carbamate (6), which was deprotected under acidic condition to obtain N-(2-((3-aminopropyl)amino)-4-fluorophenyl)-4-(((7-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)methyl)benzamide trifluoroacetate (7). Condensation of compound 7 and biotin got the target compound biotin-CN27.
Chemoproteomics
Cells were lysed in lysis buffer containing 50 mM Tris-HCI (pH 7.5), 150 mM NaCl, 2 mM EDTA, 0.3 % NP40, 1 mM PMSF on ice for 30 min. The cell lysate was then centrifuged at 13000 x g for 15 min and the supernatant was collected. The DynabeadsTM MyOneTM Streptavidin C1 were washed 3 times using wash buffer containing 5 mM Tris-HCI (pH 7.5), 1 M NaCl, 0.5 mM EDTA, 0.05 % Tween 20, and incubated at 4 °C overnight with biotin-CN27 using biotin as vehicle. The biotin pull-down assay was performed by adding the beads to 500 μL supernatant and incubated at RT for 1 h, and the beads were then washed for 3 times using 50 mM EDTA followed by 3 times using a wash buffer. Proteins were eluted using 50 µL 1×SDS-PAGE loading buffer at 95 °C for 5 min and analyzed by Coomassie brilliant blue dye and western blot.
Structure and purity verification of ZR1, ZR4, CN25, CN27 and CE7
1H NMR spectra was recorded on a Bruker DRX spectrometer at 400 MHz, with δ given in parts per million (ppm) and J in hertz (Hz) and using TMS an internal standard. Multiplicity of 1H NMR signals was reported as singlet (s), doublet (d), triplet (t), quartert (q), and multiplet (m). LC-MS was recorded on a Thermo Fisher LCF-10685 instrument equipped with an ESI source in positive ion mode. The HPLC analysis was conducted using Agilent ChemStation for LC and LC/MS Systems equipped with Synergi Hydro-RP 4 μm C18 analysis column (250 mm×4.6 mm) and UV detector.
ChIP-Seq data processing
Sequencing quality control and adapter removal for sequencing reads were performed by FastQC (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/) and fastp3, and then mapped to human reference genome hg19 using Bowtie 24. Mapped bam files of immunoprecipitations and inputs were used to call peaks by using MACS5. deepTools was used to obtain the aggregated signals of ChIP-seq across the gene bodies6.
rDNA ChIP-Seq signal analysis
Sequencing reads of immunoprecipitations and inputs were mapped to human ribosomal DNA complete repeating unit (GeneBank: U13369.1) using bowtie2 as previous study7. Because 150 bp×2 paired-end sequencing reads are long enough to distinguish repetitive elements on ribosomal DNA from whole genome, it is not necessary to map sequencing reads to the combination of human reference genome and ribosomal reference sequence. The mapped bam files were converted to bedgraph files using deeptools bamCoverage without any normalization. ChIP enrichment on ribosomal DNA was adjusted by immunoprecipitation/input of whole genome level6.
RNA-Seq data processing
Sequencing reads were mapped to human reference genome hg19 using STAR8. Mapped bam files were used to calculate FPKM using StringTie9. Gene counts were feed into DESeq2 to call differentially expressed genes10. Reproducibility of replicates was examined by hierarchical clustering using top 1000 high variation genes. Gene ontology enrichment and gene set enrichment analysis were performed by clusterProfiler and Metascape11,12. To identify downstream transcription factors potentially regulating up-regulated genes by H1-10, HOMER (finding motifs within gene promoters), TRRUST and Lisa were used13-15. We also leveraged the dataset of DepMap CCLE to investigate the genes highly correlated with H1-10, top 500 high correlated genes (Pearson correlation) were chosen to perform gene ontology enrichment and transcription factor target analysis using Metascape12,16.
Protocol references
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