May 03, 2024

Public workspaceMime-seq 2.0: a method to sequence microRNAs from specific mouse cell types V.1

DOI
dx.doi.org/10.17504/protocols.io.rm7vzxzzxgx1/v1
  • 1Research Institute of Molecular Pathology (IMP);
  • 2School of Medicine, John Hopkins University;
  • 3Max Perutz Labs (MPL);
  • 4University of Vienna
Ariane Mandlbauer
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DOI: dx.doi.org/10.17504/protocols.io.rm7vzxzzxgx1/v1
Protocol Citation: Ariane Mandlbauer, Qiong Sun, Niko Popitsch, Tanja Schwickert, Miroslava Spanova, Jingkui Wang, Stefan Ameres, Meinrad Busslinger, Luisa Cochella 2024. Mime-seq 2.0: a method to sequence microRNAs from specific mouse cell types. protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vzxzzxgx1/v1Version created by Ariane Mandlbauer
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: February 23, 2024
Last Modified: May 03, 2024
Protocol Integer ID: 95667
Keywords: miRNAs, miRNA sequencing, miRNA methylation, miRNA profiling, rare cell types, mammalian system, B cells
Funders Acknowledgements:
Luisa Cochella
Grant ID: NSF CAREER Award 2238425
Stefan Ameres
Grant ID: ERC-CoG-866166
Stefan Ameres
Grant ID: SFB-F8002
Abstract
The description of precise miRNA expression patterns is crucial to understand what these small RNAs contribute to animal development and physiology. High-throughput sequencing of miRNAs from individual developmental stages has provided insight into temporal regulation but often lacks the cellular resolution to link miRNA-function to the biology of distinct cell types within complex tissues. Here, we provide a protocol for microRNome by methylation-dependent sequencing for mice (mime-seq 2.0). Mime-seq takes advantage of a chemical small RNA-tagging approach followed by chemo-selective, high-throughput sequencing that enables the identification of tissue- and cell type-specific miRNA profiles in animals in a sensitive and reproducible manner.
Materials
Reagents
NameSupplierCatalogue number
2-propanolFisherChemicalC298-500
GlycogenSigmaG1767-1VL
EtOH abs.Avantor20821.321
H2O, DEPC-treated InvitrogenAM9920
H2O, Rnase freeInvitrogenAM9935
Qubit RNA BR Assay KitInvitrogenQ10210
Ammonium PersulfateSigmaA3678
TEMEDSigmaT9281
Sodium PeriodateSigma311448-5G
Sodium Acetate (3M) ph 5.5InvitrogenAM9740
NaOHSigmaS5881
Boric acidSigmaB6768
Sodium tetraborateSigma311448-5G
Sodium chloride Sigma106404
SequaGel UreaGel SystemNational DiagnosticsEC-833
Gel Loading Buffer IIInvitrogenAM8547
Hybond-NX membraneCytivaRPN303T
MethylimidazoleSigmaM50834
EDCSigmaE7750-25G
EDTA (0.5M) pH 8Invitrogen15575020
Sodium Phosphate dibasic heptahydrateSigma431478
Phosphoric AcidAvantor02-003-602
T4 Polynucleotide KinaseNew England BiolabsM0201L
g-32P-ATPPerkin ElmerBLU002Z250UC
MicroSpin G-25 ColumnsCytiva27532501
Storage Phosphor ScreenCytiva28956474
Protein G Dynabeads Invitrogen10003D
K227Q truncated T4 RNA ligase 2 New England BiolabsM0351L
PEG8000 50%New England BiolabsB1004A
SYBR Gold InvitrogenS11494
TBE buffer, 10xQuality Biological351-001-131
SSC buffer, 20xSigmaS6639-1L
T4 RNA ligase 1 New England BiolabsM0204L
Superscript II or III reverse transcriptase Invitrogen18064014/18080044
RNaseOUTInvitrogen10777019
ExoSAP-IT Applied Biosystems78201
Kapa HiFi HotStart Library Amp kit RocheKK2612
Zymoclean Gel DNA recovery kit ZymoD4008
Low Range AgaroseBioRad1613107
IGEPAL CA-630SigmaI3021
Sodium DeoxycholateSigmaD6750
Sodium Dodecyl Sulfate SigmaL3771
Sodium Dodecyl Sulfate 20% solutionSigma05030
TrisThermo Scientific75825
GlycerolSigma15523
Bromophenol BlueBioRad1610404
2-Mercaptoethanol (BME)SigmaM-6250
TweenSigmaP9416
Magnesium ChlorideAvantor7791-18-6

Plasticware
  • DNA LoBind tubes 1.5 mL (Eppendorf, G172215J)
  • Standard transparent heavyweight 
sheet protector
  • Optical Flat 8-Cap strips (BioRad, TCS0803)
  • Individual PCR Tubes 8-Tube strip, clear (BioRad, TLS0801)

Equipment
NameCompany
Typhoon phosphorimager Cytiva
HiSeq V4 sequencer or similarIllumina
Qubit flex fluorometerInvitrogen
Blue light transilluminator
Gel electrophoresis apparatus vertical and horizontal
ChemiDocBioRad
(optional) fragment analyzer or similarAgilent
Buffer recipes
  • 5x Borate Buffer 300 mM, pH 8.6
Dissolve 5.49 g Sodium tetraborate decahydrous (Na2B4O7:10H2O[LYC1] , MW = 381.37 g/mol) in 80 ml H2O, fill up to 100 ml; dissolve 2.814 g Boric acid (H3BO3, MW = 61.83 g/mol) in 80 ml H2O, fill up to 100 ml. Mix equal volumes (i.e. 50 ml + 50 ml) to achieve 300 mM solution, adjust pH to 8.6.

  • PAGE elution buffer (0.3 M NaCl, 0.1% SDS)

  • 6x Orange G loading dye (4 g /l orange G, 40% sucrose)

  • 10x 3’ ligation buffer (500 mM Tris pH 7.8, 100 mM MgCl2, 100 mM DTT)

  • 10x T4 Ligase Buffer (500 mM Tris pH 7.8, 100 mM MgCl2, 10 mM ATP 100 mM DTT)

Sequences
Download sequences.xlsxsequences.xlsx


Total RNA isolation
Total RNA isolation
40m
40m
Extract total RNA from frozen cells or tissue using TRIzol, depending on sample type follow manufacturer’s instructions for proper lysis. 
Critical
Add 200 μl of chloroform per 1 ml of TRIzol, agitate samples vigorously and incubate for 5 min at room temperature (RT).
5m
Centrifuge at full speed (12,000 x g) for 15 min at 4°C. The mixture separates into lower, red, phenol-chloroform phase, an interphase, and a colorless aqueous phase. 
15m
Centrifigation
Carefully transfer the upper aqueous phase to a fresh tube.
Precipitate RNA by adding 1 μl of glycogen (20 mg/ml) and 500 μl of isopropyl alcohol, vortex.
Incubate at 4˚C for 10 minutes.
10m
Incubation
Centrifuge at full speed for 10 minutes at 4˚C; RNA will form a gel-like pellet on the side and bottom of the tube; for low input samples, extend centrifugation time to 60 min.
10m
Centrifigation
Temperature
Remove supernatant without losing the RNA pellet.
Critical
Wash pellet
Wash
Add 300 μl of 80% cold ethanol, vortex.
Centrifuge 10 minutes at full speed at 4°C.
Centrifigation
Temperature
Remove supernatant, collect remaining ethanol by quick centrifugation and pipette all remaining liquid using a 10 μl pipette. Air dry pellet for approx. 5 min or until it becomes translucent.
Air dry pellet for approx. 5 min or until it becomes translucent.
Dissolve RNA pellet in 20-50 μl of DEPC-treated or other RNase free water, to obtain a target RNA concentration above 1 μg/μl.
Measure concentration (Qubit BR RNA kit); ideally, at least 3.3 μg of total RNA are used for small RNA library preparation per sample (will be split in ox/unox).
NaIO4-mediated oxidation
NaIO4-mediated oxidation
30m
30m
Prepare 3.3 μg of total RNA with a mix of methylated and unmethylated spike-ins in 44 μl RNase-free H2O (includes 10 % extra).
For Oxidation reaction, split each sample in half and incubate using following reaction mix 30 min at RT.
ReagentVolume
RNA/Spike-in mix20 ul
NaIO4 (50mM) or H2O5 ul
5x borate buffer8 ul
H2O7 ul
Prepare a master mix +/- NaIO4 containing everything except for the respective sample. Transfer samples for oxidation and control reaction in 8 strips, distribute master mix last.

30m
Incubation
Critical
Add immediately 229 ul H2O, 30 ul NaOAc (pH 5.2) and 1 ul glycogen (20 mg/ml), mix well.
EtOH precipitation
EtOH precipitation
Precipitate by adding 900 ul ice-cold EtOH to each sample.
Incubate for > 1h at -20˚C (this can be overnight).
1h
Incubation
Pause
Temperature
Centrifuge for 1 h at 4˚C and remove supernatant without disturbing the pellet.
1h
Centrifigation
Critical
Temperature
Wash
Add 500 μl of 80% cold ethanol.
Centrifuge 10 minutes at full speed at 4°C.
Remove supernatant, collect remaining ethanol by quick centrifugation and pipette all remaining liquid using a 10 μl pipette.
Air dry pellet for approx. 5 min or until it becomes translucent.
Resuspend each pellet in 6 ul H2O (DEPC-treated or RNase free).
3' Ligation
3' Ligation
Add to each sample 1 μl srBC 3’ adapter (10 μM) and note used barcodes.

Note – oxidized samples should not be run on the same gel as unoxidized samples, due to big differences in ligatable small RNAs. Each sample run on the same gel should have a different srBC adapter. 

Set up in parallel ligation reaction for 18mer and 30mer as later reference for PAGE purification. Mix 2.5 μl 18mer (10 μM) with 2.5 μl 30mer (10 μM) and 0.5 μl 100 μM 3’ linker and 1.5 μl H2O.

Thaw 50% PEG8000 at 37˚C, this makes pipetting of viscous solution easier. 
Incubate RNA + srBC 3’ 5 min at 70˚C, snap-cool on ice.
       Set up master mix at room temperature:
ReagentVolume
PEG8000 50%10 ul
10x 3' Ligation buffer2 ul
T4 RNA ligase trunc2 ul

Add 13 µl MM to each sample and incubate o/n at 4˚C.

Note – we normally keep the ligation reactions in a metal cooling rack in the fridge or cold room.
Incubation
Overnight
Temperature
Size selection (PAGE)
Size selection (PAGE)
Prepare a 15% denaturing urea polyacrylamide gel (SequaGel; 20 cm × 16 cm × 1 mm, length × width × thickness).

Note – pre-treat glass plates (one with anti-fog, the other with water repellent) to avoid gel sticking to the plates.
Pre-run the gel in 0.5× TBE buffer Tat a constant power of 35 W for 30-60 min until the surface temperature of the gel reaches approximately 45–55°C.
In the meantime add an equal volume of form amide loading buffer to the samples and denature by incubating 5 min at 95ºC, snap-cool on ice.

Note – include ligated 18 and 30mers, as well as unligated 60mer as size reference.
Load samples into pre-washed wells.

Note – IMPORTANT rinse all wells before loading with a syringe to remove all accumulated urea and gel fragments. Rinse again before loading of each sample. 

Do not use lanes with bubbles and leave at least one empty lane between each sample for easier handling. Do not run oxidized and unoxidized samples on the same gel. 
Run gel first at constant power of 10 W until samples are migrated out of their wells. Run at a constant power of 35 – 45 W until the bromophenol blue is about halfway down the gel (1-1.5 h).
Remove the top glass plate and with the gel still on the bottom plate, pour 10-15 ml of SYBR gold in 0.5x TBE (for 50 ml use 5 μl of SYBR gold) and stain for 3-4 minutes.
Carefully transfer the gel onto a transparent sheet protector .

Note – open sheet protector on 3 sides, hold gel on one end and transfer between sheets. We normally use tape on the outside to label the individual sample lanes for orientation. 
Visualize RNA on blue transilluminator (take a photograph if desired) and label area between ligated 18mer and 30mer on each lane on the sheet protector with a marker. Remove from transilluminator and cut out using a razor blade small RNAs between markers, collect in 1.5 ml LoBind tubes.

Note – cutting the gel piece in half and laying them on top of each other makes it easier to transfer gel piece to a tube.
Add 800 µl 0.3 M NaCl + 0.1% SDS and allow RNA to elute overnight at room temperature, while rotating.
Overnight
EtOh precipitation
EtOh precipitation
Precipitate each sample by transferring to two fresh tubes (without transferring gel pieces) and adding to each 400 µl aliquot, 1 µl glycogen (20 mg/mL) and 1 ml of 100% EtOH; incubate samples for 1.5-2 hours at -20˚C.
Centrifuge 40 minutes at full speed (12,000 x g) at 4˚C, decant supernatant and wash the pellet with 300 µl of 80% cold ethanol as before. Centrifuge again 10 minutes at 4˚C, remove the supernatant completely, perform an extra round of centrifugation for 2 minutes and pipette out all residual EtOH.
Without air-drying the pellet, dissolve small RNAs in 6 µl H2O + 1 µl 5’ adapter (10 µM).
5’ linker ligation
5’ linker ligation
Incubate RNA + adapter mix 5 min at 70˚C, snap-cool on ice.
Prepare ligation master mix (volumes are calculated for one sample):

ReagentVolume
PEG8000 50%10 ul
10x RNA ligae buffer2 ul
T4 RNA Ligase 12 ul

Add to each sample 13 µl of master mix, mix and give it a quick spin.
Incubate o/n at 4˚C.

Note – we normally keep the ligation reactions in a metal cooling rack in the fridge or cold room.
Incubation
Overnight
Temperature
Purification using RNA Clean & Concentrator 5 (Zymo)
Purification using RNA Clean & Concentrator 5 (Zymo)
Add 80 µl H2O and mix by pipetting.
Purify using the RNA Clean & Concentrator 5 (RCC5) kit
Add 200 µl RNA binding buffer and mix.
Add 300 µl EtOH absolute and mix.
Continue according to manufacturer's instructions and elute in 12 µl H2O.
Reverse transcription
Reverse transcription
Transfer 11.5 µl sample into a PCR strip and add 1 µl RT primer (10 µM).
Incubate 5 min at 65˚C, snap-cool on ice.

Note – pre-heat PCR block to 50˚C.
RT reaction master mix:
ReagentVolume
5x FS buffer4 ul
dNTP mix (10mM)1 ul
0.1 M DTT1 ul
RNase out0.5 ul
Superscript II or III1 ul

Add on ice 7.5 µl RT master mix, mix well, spin down and immediately transfer to pre-heated PCR block at 50˚C, incubate 30 min.

Note – IMPORTANT, no heat inactivation step after RT.
Add 8 µl ExoSAP-IT and incubate 15 min at 37˚C followed by heat inactivation at 80˚C for 15 min.
On ice, add 2 µl H2O for a total volume of 30 µl.
qPCR amplification
qPCR amplification
Transfer ½ of each sample (15 µl) to strip tube. Add 10 µl i5/i7 primer mix and 25 µl Kapa HiFi Library Amp Kit mix.
Prepare also in duplicates 50 µl of fluorescent standard (STD) 3 and 4.
Close strip with an optical flat-cap, vortex, and spin down.

Run the following program until samples reach the fluorescence level of standard 3 (assessed from the image acquired by the PCR machine in each cycle).

Note - use qPCR machine (i.e. BioRad CFX96) to monitor library amplification in real-time. Base-line subtraction hould be turned off.

TemperatureDurationRepetitions
95˚C45 sec
95˚C15 secn cycles
65˚C15 sec
72˚C20 sec
plate read
72˚C10 sec
When a sample reaches signal between standard 3 and 4, pause the program during the 10 sec step after the plate read and remove carefully the respective tubes from the PCR machine. Repeat until all samples are amplified sufficiently.


Figure 1: Example of real-time qPCR amplification plot for unoxidized and oxidized samples. Each sample requires a certain number of cycles (x axis) to reach the desired amount (fluorescence level in y axis), depending on the amount of starting material. Unoxidized samples (-) indicated in light green, oxidized samples (+) in dark green. fluorescent STD 3 in pink and STD 4 in blue.


PCR
Gel purification of amplified cDNA
Gel purification of amplified cDNA
Prepare a 3% Low-Range Ultra agarose gel according to the manufacturer’s instructions (swirl the flask often during heating procedure), with 1.5 µl SYBR Safe per 50 ml of gel.
Load 6 µl of GeneRuler 50 bp DNA Ladder and the amplified cDNA after addition of 10 µl of Orange G loading dye 6x (total volume 60 µl)
Run the gel at constant V (depending on chamber size between 80 and 150V) until the Orange G loading dye band is at 2/3 of the gel length.
Visualize on a UV transilluminator and excise upper DNA band between 160-200 bp with a clean scalpel or razor blade and place in a clean tube, as shown in Figure 2 below.

Note - IMPORTANT in case 2 bands are visible – avoid cutting into lower band as this corresponds to adapter dimers; this is especially common with oxidized library samples.


Figure 2: Example of 3% low-range agarose gel, before and after DNA band excision. Oxidized samples 1-4 were loaded on the left gel using the GeneRuler 50 bp DNA Ladder as reference, unoxidized samples are loaded on the right gel. Please, notice that the desired upper band can be very faint in case of oxidized sample (i.e. 1+). Even in those cases, cut above the strong band of adaptor dimers and proceed with DNA extraction (see below).

Dissolve gel by adding 3 volumes (~800-900 µl) of ADB buffer (ZymocleanTM Gel DNA Recovery Kit) and incubating samples at 55ºC for ~10 minutes or until the gel slice is completely dissolved; mix and collect by centrifugation.
Transfer the melted agarose solution to a Zymo-SpinTM Column in a collection tube, centrifuge 1 minute at 10,000 rpm and discard the flow-through.
Add 200 µl of DNA Wash Buffer to the column, centrifuge 1 minute and discard the flow-through; repeat the wash step once more.
Add 20 µl of water directly to the column matrix, place the column into a 1.5 ml tube and centrifuge for 1 minute to elute DNA; purified DNA is now ready for quality control and deep sequencing.

Note - we routinely run libraries on fragment analyzer or tape station to make sure adapter dimers are sufficiently removed.
Bioinformatics and data analysis
Bioinformatics and data analysis
Libraries are sequenced using an Illumina short read sequencer with a minimum read length of 50 nucleotides as for example NovaSeq SP in SR100 XP mode.
Custom NextFlow pipelines for processing can be found on GitHub under https://github.com/popitsch/pysrna. In short:

Sequencing quality control with fastqc v 0.11.8. 
Raw reads are demultiplexed, parsed and filtered.
Spike-in reads are filtered and counted allowing for alignment 1 mismatch.
Remaining small RNA reads are mapped to a miRNA transcriptome obtained from MirGeneDB v2.0 or miRbase annotations.
Small RNAs were counted and normalized to reads obtained from methylated spike-ins.