Sep 30, 2022
Version 2
METATAC V.1 V.2
- Honggui Wu1,
- Xiang Li2,1,3,
- Fanchong Jian2,1,4,
- Ayijiang Yisimayi2,1,5,
- X. Sunney Xie2,1,6
- 1Biomedical Pioneering Innovation Center, Peking University, Beijing 100871, China;
- 2Beijing Innovation Center for Genomics, Peking University, Beijing 100871, China;
- 3Peking-Tsinghua Center for Life Sciences, Academy for Advanced Interdisciplinary Studies, Peking University, Beijing 100871, China;
- 4College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, PRC;
- 5School of Life Sciences, Peking University, Beijing, 100871, China;
- 6School of Life Sciences, Peking University, Beijing 100871, China Peking-Tsinghua Center for Life Sciences, Academy for Advanced Interdisciplinary Studies, Peking University, Beijing 100871, China
External link: https://www.pnas.org/doi/10.1073/pnas.2206450119
Protocol Citation: Honggui Wu, Xiang Li, Fanchong Jian, Ayijiang Yisimayi, X. Sunney Xie 2022. METATAC V.1. protocols.io https://dx.doi.org/10.17504/protocols.io.kqdg36x51g25/v2Version created by Honggui Wu
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: September 30, 2022
Last Modified: September 30, 2022
Protocol Integer ID: 70695
Keywords: scATAC-seq, chromatin accessibility, single-cell epigenomics, meta transposome, cell chromatin accessibility analysis, accessible chromatin, chromatin accessibility analysis, maximum dna recovery, tn5 from bound dna, cell atac, metatac, nextera transposome, bound dna, meta chemistry, dna, meta sequence, atac protocol, extensive biochemical modification, tagging amplification,
Funders Acknowledgements:
the Beijing Municipal Commission of Science and Technology
Grant ID: Z201100005320015
Abstract
Abstract
Here we describe a protocol for multiplexed end-tagging amplification of transposase accessible chromatin (METATAC), a high-sensitivity single-cell ATAC-seq technique with the help of META chemistry and extensive biochemical modifications. We improved the protocol from three aspects, first, we used Omni-ATAC protocol to permeabilize cells, which greatly reduced mitochondrial reads to less than 0.5%. Second, we used sodium dodecyl sulfate (SDS) to release Tn5 from bound DNA, which enabled maximum DNA recovery. Third, we use META transposome instead Nextera transposome used in other protocols, which avoids half loss due to self-looping during amplification, thus further increasing library size. Specifically, META sequences also serve as a barcode to fragment decontamination, which provides high-sensitivity and precise single-cell chromatin accessibility analysis.
Protocol materials
IGEPAL CA-630Merck MilliporeSigma (Sigma-Aldrich)Catalog #I8896-50ML
Tween 20Bio-Rad LaboratoriesCatalog #170-6606-MSDS
Digitonin, 40ulPromegaCatalog #G9441
NN-Dimethylformamide anhydrousMerck MilliporeSigma (Sigma-Aldrich)Catalog #D4551-250ML
Troubleshooting
REAGENT SETUP
1h 30m
Prepare METATAC Reagents
1h 30m
5 mL ATAC resuspension buffer (ATAC-RSB)
Mix 50 µL 1 M Tris-HCl pH 7.5, 10 µL 5 M NaCl, 15 µL 1 M MgCl2, and bring the final volume to 5 mL with nuclease-free H2O. Store the buffer at -20 °C for up to several months.
| A | B | C | |
| Reagents | 5 mL | Final conc. | |
| 1 M Tris-HCl pH 7.5 | 50 µL | 10 mM | |
| 5 M NaCl | 10 µL | 10 mM | |
| 1 M MgCl2 | 15 µL | 3 mM | |
| Nuclease-free H2O | 4925 µL | ||
| Total | 5 mL |
200 µL Omni-ATAC lysis buffer
50 µL for each reaction, mix 200 µL ATAC-RSB with 2 µL 10% IGEPAL CA630IGEPAL CA-630Merck MilliporeSigma (Sigma-Aldrich)Catalog #I8896-50ML ,2 µL 10 Tween 20Tween 20Bio-Rad LaboratoriesCatalog #170-6606-MSDS , 2 µL 1% digitoninDigitonin, 40ulPromegaCatalog #G9441 . Freshly prepare before use.
| A | B | C | |
| Reagents | 200 µL | Final conc. | |
| ATAC RSB | 200 µL | ||
| 10% IGEPAL CA630 | 2 µL | 0.1% | |
| 10 % Tween 20 | 2 µL | 0.1% | |
| 1% Digitonin | 2 µL | 0.01% |
600 µL Omni-ATAC wash buffer
150 µL for each reaction, mix 600 µL ATAC-RSB with 6 µL 10% Tween 20. Freshly prepare before use.
| A | B | C | |
| Reagents | 600 µL | Final conc. | |
| ATAC RSB | 600 µL | ||
| 20% Tween 20 | 6 µL | 0.1% |
1 mL 2 x TD buffer
mix 20 µL 1 M TAPS pH 8.5, 10 µL 1 M MgCl2, 200 µL DMFNN-Dimethylformamide anhydrousMerck MilliporeSigma (Sigma-Aldrich)Catalog #D4551-250ML , and bring the final volume to 1 mL with nuclease-free H2O. Store the buffer at -20 °C for up to several months.
| A | B | C | |
| Reagents | 1 mL | Final conc. | |
| 1 M TAPS pH 8.5 | 20 µL | 20 mM | |
| 1 M MgCl2 | 10 µL | 10 mM | |
| DMF | 200 µL | 20% | |
| Nuclease-free H2O | 770 µL |
1 mL 2 x STOP buffer
mix 80 µL 0.5 M EDTA, 10 µL 1 M Tris-HCl pH 8.0, 10 µL 0.1M spermidine, and bring the final volume to 1 mL with nuclease-free H2O. Freshly prepare before use.
| A | B | C | |
| Reagents | 1 mL | Final conc. | |
| 0.5 M EDTA | 80 µL | 40 mM | |
| 1 M Tris-HCl pH 8.0 | 10 µL | 10 mM | |
| 0.1 M spermidine | 10 µL | 1 mM | |
| Total | 1 mL |
50 µL Assemble META16 transposome
METATAC_Primer_v.1.xlsx
METATAC_Primer_v.1.xlsx 1) Anneal META16 transposon
| A | B | |
| Oligos | Sequence | |
| META16-1 | GGCACCGAAAA | |
| META16-2 | CTCGGCGATAAA | |
| META16-3 | GGTGGAGCATAA | |
| META16-4 | CGAGCGCATTAA | |
| META16-5 | AGCCCGGTTATA | |
| META16-6 | TCGGCACCAATA | |
| META16-7 | GCCTGTGGATTA | |
| META16-8 | GCGACCCTTTTA | |
| META16-9 | GCATGCGGTAAT | |
| META16-10 | GCGTTGCCATAT | |
| META16-11 | GGCCGCATTTAT | |
| META16-12 | ACCGCCTCTATT | |
| META16-13 | CCGTGCCAAAAT | |
| META16-14 | TCTCCGGGAATT | |
| META16-15 | CCGCGCTTATTT | |
| META16-16 | CTGAGCTCGTTTT | |
| 19 bp ME | 5'-/phos/-CTGTCTCTTATACACATCT-3' | |
| META Tranposon | 5'-[META sequence]-AGATGTGTATAAGAGACAG-3' |
| A | B | C | |
| Reagents | Per 50 µL | Final conc. | |
| 10 x Annealing Buffer | 5 µL | 1x | |
| 50 µM META16 Transposon | 5 µL | 5 µM | |
| 50 µM 19 bp ME | 5 µL | 5 µM | |
| H2O | 35 µL |
Mix thoroughly, then run the annealing program (95 °C, 1 min, gradual cooling, -0.1 °C /3s, 700 cycles to 25 °C, hold at 4 °C)
Recipe for 10x annealing buffer (500 mM NaCl, 100 mM Tris-HCl pH 8.0, 10 mM EDTA)
2) Assemble METAT16 transposome
Mix 25 µL 5 µM Tn5 transposase and 25 µL 5 µM annealed META16 transposon, incubate at 21-24 °C for 30 min, protected from light.
Note
Assembled transposome can be stored at -80 °C for up to six months. Tn5 transposase was expressed in-house from the plasmid pTXB1-Tn5 or purchased from Vazyme (s111-01).
Assembled META16 transposome at a final concentration of 1.25 µM dimer.
Bulk Transposition
1h 30m
Harvest fresh culture in a conical centrifuge tube (15 ml or 50 ml) at room temperature, centrifuge at 500 x g for 5 min at room temperature, then wash twice with 1x PBS pH 7.4, count cell number, stain with Trypan blue, and ensure viability >90%. then aliquot 50, 000 cells to a 200 µL PCR tube.
Note
In order to have enough nuclei for FACS, we recommend preparing 2-3 replicates, which is enough for 50-60 96-well plates.
Note
Our protocol also works well for cryopreserved samples and nuclei. For the cryopreserved sample, quickly thaw one tube of the cell at 37 °C water bath, then wash once with ice-cold PBS, count cell number, aliquot 50,000 each.
20m
Pellet 50,000 viable cells at 500 x g at 4°C for 5 min in a swing bucket centrifuge, and remove supernatant carefully without disturbing the pellet.
6m
Add 50 µL ice-cold Omni-ATAC lysis buffer (step 1.2), pipette up and down 10 times, then incubate on ice for 3 min.
Note
The incubation time depends on cell type, 3 min works well for most samples, but we found for the digestive system sample, incubation needs to extend to 5-10 min to get the periodic nucleosomal pattern.
4m
Wash out lysis with 150 µL of ice-cold Omni-ATAC wash buffer (step 1.3) and invert the tube 3 times to mix.
2m
Pellet nuclei at 500 x g for 10 min at 4°C in a swing bucket centrifuge.
11m
Then wash one time with 50ul ice-cold Omni-ATAC wash buffer. Pellet nuclei at 500 x g at 4°C for 5min.
8m
Transposition in Bulk
50m
Prepare Transposition mix
| A | B | C | D | |
| Reagents | 3 Rxn | Per Rxn | Final conc. | |
| 2 x TD buffer | 37.5 µL | 12.5 µL | ||
| META 16 Transposome | 6 µL | 2 µL | 100 nM | |
| 1 x PBS | 28.5 µL | 9.5 µL | ||
| 1% Digitonin | 0.75 µL | 0.25 µL | 0.01% | |
| 10% Tween 20 | 0.75 µL | 0.25 µL | 0. 1% |
Aspirate all supernatant, and avoid disrupting the visible pellet. Then resuspend the cell pellet in 25 µL of transposition mixture by pipetting up and down 10 times, then transfer to a 1.5 mL Lo-bind tube.
Incubate the reaction at 37°C for 30 minutes in a thermomixer with 1000 RPM mixing.
Add 25 µL 2x Stop buffer to stop transposition and incubate on ice for 10 min.
FACS single nuclei
1h
Add 50 µL 0.5% BSA (by dissolving 0.25 g BSA in 50 mL 1x PBS pH 7.4), then add 5 µL 7-AAD to stain nuclei.
FACS sort single 7-AAD positive nuclei to a 96-well PCR plate, containing 1 µL nuclei lysis buffer (10 mM Tris-HCl pH 8.0, 20 mM NaCl, 1 mM EDTA pH 8.0, 15 mM DTT, 0.1% SDS, 60 µg/mL QIAGEN protease).
Note
If not proceed immediately, seal the plate with Aluminum Sealing Film (Axygen, PCR-AS-600), and store at -80°C for several months without lysis.
Note
All liquid transfer steps can be done with a multichannel pipette or with an automated liquid handler system (e.g., Beckman Biomek FXP liquid handler, Echo 525 acoustic liquid handler system). Here, we use Echo 525 to handle all the liquid transfer steps, which process 20 plates each time.
For cell lysis buffer, we use Echo 525 to aliquot with 384PP_AQ_BP calibration, 45 s/plate.
Amplification
3h 30m
Seal the plate with PCR sealing film (bio-rad, MSB1001), lysis was done by incubating at 65 °C for 15 min.
30m
After lysis, add 1 µL 3% Triton X-100 to quench SDS. Spin down in a plate centrifuge, vortex to mix.
Note
For 3% Triton X-100, we use Echo 525 to aliquot with 384PP_AQ_SPHigh calibration. 45 s/plate.
20m
Amplification
2h
Prepare preamplification mix
| A | B | C | D | |
| Reagents | 120 Rxn | Per rxn | Final conc. | |
| 2 x High fidelity Q5 master mix | 360 | 3 | 1x | |
| 50 µM META16 primer mix | 23.04 | 0.192 | 100 nM each | |
| 100 mM MgCl2 | 6 | 0.05 | ||
| Nuclease-free H2O | 90.96 | 0.758 | ||
| Cell lysate | NA | 2 | NA |
| A | B | |
| Oligos | Sequence | |
| META 16 Primer | 5'-[META sequence]-AGATGTGTATAAG-3' |
Note
META16 primer sequences see above .
20m
Aliquot 4 µL above preamplification mix to each well, Spin down in a plate centrifuge, vortex to mix.
Note
For the preamplification mix, we use Echo 525 to aliquot with 384PP_AQ_BP calibration, 2 min/plate.
45m
Preamplification was incubated as
72°C, 5 min
98°C, 30 s
16 Cycles [98°C , 10 s; 62°C, 30 s; 72°C, 1 min]
72°C, 5 min
4°C hold.
50m
Cell barcoding
Note
Cell barcoding was realized using a 12 x 8 barcode combination. Premix 12 META16-ADP1 barcodes and 8 META16-ADP2 barcodes into 96 barcode combinations. Detailed sequences see below table.
| A | B | |
| Oligos | Sequence | |
| META16-ADP1 | 5'-CTTTCCCTACACGACGCTCTTCCGATCT-[CB1]-[META sequence]-AGATGTGTATAAG-3' | |
| META16-ADP2 | 5'-GAGTTCAGACGTGTGCTCTTCCGATCT-[CB2]-[META sequence]-AGATGTGTATAAG-3' | |
| CB1-1 | GATATG | |
| CB1-2 | ATACG | |
| CB1-3 | CCGTCTG | |
| CB1-4 | TGCG | |
| CB1-5 | GAACTCG | |
| CB1-6 | ATGTAG | |
| CB1-7 | CCCG | |
| CB1-8 | TATGT | |
| CB1-9 | GAGTAAG | |
| CB1-10 | ATCG | |
| CB1-11 | CCTAG | |
| CB1-12 | TGACCG | |
| CB2-1 | ACTCTA | |
| CB2-2 | AGAGCAT | |
| CB2-3 | GGTATG | |
| CB2-4 | TCGATGC | |
| CB2-5 | CTACTAG | |
| CB2-6 | TATGCA | |
| CB2-7 | CACACGA | |
| CB2-8 | GTCGAT | |
Add 0.45 µL of one of 96 barcode mixes to each well.
Incubate as
98°C, 30 s,
5 cycles [98°C, 10 s, 62°C, 30 s, 72°C, 1 min]
72°C, 5 min
Note
For the cell barcode, we use Echo 525 to aliquot with 384PP_AQ_BP calibration, 30 s/plate.
40m
Pool a whole plate for purification, typical 200 µL/plate for purification. DNA was extracted with ZYMO DCC5.
It's a typical amplicon of GM12878 cells, but the fragment size distribution varies according to different cell types.
1h 30m
For fragment analysis, we use Agilent Fragment analyzer DNF474 kit, only samples with clear periodic nucleosome patterns are used for sequencing.
Wxamplified library showed good periodic nucleosome pattern.
Examplified library showed poor periodic nucleosome pattern.
Library preparation
Library Preparation
1h 30m
Prepare Library prep mix
| A | B | C | D | |
| Reagents | 40 Rxn | Per rxn | Final conc. | |
| 2x Q5 master mix | 600 | 15 | 1x | |
| NEB universal primer(10 µM) | 20 | 2 | 0.67 µM | |
| Neb i7 Index primer(10 µM) | 2 | 0.67 µM | ||
| 100 mM MgCl2 | 1 | 0.1 | ||
| Template | 10.9 | |||
| Total | 30 |
Library preparation was done by incubating as
98°C, 30 s
2 cycle [98°C, 10 s, 68°C, 30 s, 72°C, 1 min]
72°C, 5 min
Note
2 cycles of amplification are critical to avoid residual cell barcode primers in purified amplicons causing cell-to-cell contamination.
Purify with ZYMO DCC5, then purify with 1.1 x SPRI beads to remove primer dimers.
METATAC Library schematic diagram
Sequencing
For sequencing, we sequenced our sample on Illumina Hiseq 4000 or NovaSeq sequencer with 9 Gb/plate.
Raw read processing.
Raw Read Preprocessing. For both read 1 and read 2, the first 4 to 7 bases and the following 11 to 13 bases are paired cell barcodes and META sequence, respectively (step 1.6 attachment). We used a custom Python script to parse barcodes and split reads into individual fastq files for each cell, allowing up to one mismatch. Meanwhile, META sequences were annotated to read the name, allowing up to two mismatches. Then we used cutadapt to trim adapter sequences from both ends according to the 19-bp mosaic end (ME) sequence, with parameters -e 0.22 -a CTGTCTCTTATACACATCT and -e 0.22 -g AGATGTGTATAAGAGACAG for both read 1 and read 2. Processed reads were mapped to reference genome with bowtie2 -X 2000 –local –mm –no-discordant –no-mixed. hg38 (GRCh38, v26) reference genome was used for human cells, and mm10 (GRCm38, vM19) reference genome was used for mouse cells. Reads with mapping quality of less than 30 were filtered out from the further analysis. PCR duplicates were identified and removed with a custom script, according to their positions on the genome and META tags. Paired-end reads were converted to fragments with Tn5 insertion centering correction (R1 start +4 and R2 end 5). Finally, for each cell, contaminated fragments from other cells were removed based on the aligned coordinates, META sequences, and read frequency.