TITLE: Mime-seq 2.0: a method to sequence microRNAs from specific mouse cell types
AUTHORS: Ariane Mandlbauer, Qiong Sun, Niko Popitsch, Tanja Schwickert, Miroslava Spanova, Jingkui Wang, Stefan Ameres, Meinrad Busslinger, Luisa Cochella

[DESCRIPTION]
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.

[STEPS]
SECTION: Total RNA isolation
11. 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).
SECTION: Total RNA isolation
1. Extract total RNA from frozen cells or tissue using TRIzol, depending on sample type follow manufacturer’s instructions for proper lysis. 
SECTION: Total RNA isolation
2. Add 200 μl of chloroform per 1 ml of TRIzol, agitate samples vigorously and incubate for 5 min at room temperature (RT).
SECTION: Total RNA isolation
3. 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. 
SECTION: Total RNA isolation
4. Carefully transfer the upper aqueous phase to a fresh tube.
SECTION: Total RNA isolation
5. Precipitate RNA by adding 1 μl of glycogen (20 mg/ml) and 500 μl of isopropyl alcohol, vortex.
SECTION: Total RNA isolation
6. Incubate at 4˚C for 10 minutes.
SECTION: Total RNA isolation
8. Remove supernatant without losing the RNA pellet.
SECTION: Total RNA isolation
10. 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.
SECTION: Total RNA isolation
7. 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.
SECTION: Total RNA isolation
9. Wash pellet
SECTION: Total RNA isolation
9.1. Add 300 μl of 80% cold ethanol, vortex.
SECTION: Total RNA isolation
9.3. 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.
SECTION: Total RNA isolation
9.2. Centrifuge 10 minutes at full speed at 4°C.
SECTION: NaIO4-mediated oxidation
12. 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).
SECTION: NaIO4-mediated oxidation
13. For Oxidation reaction, split each sample in half and incubate using following reaction mix 30 min at RT.
 

SECTION: EtOH precipitation
15. Precipitate by adding 900 ul ice-cold EtOH to each sample. 
SECTION: EtOH precipitation
16. Incubate for > 1h at -20˚C (this can be overnight).
SECTION: EtOH precipitation
17. Centrifuge for 1 h at 4˚C and remove supernatant without disturbing the pellet. 
18. Wash
SECTION: NaIO4-mediated oxidation
14. Add immediately 229 ul H2O, 30 ul NaOAc (pH 5.2) and 1 ul glycogen (20 mg/ml), mix well.
18.1. Add 500 μl of 80% cold ethanol.
18.2. Centrifuge 10 minutes at full speed at 4°C.
18.3. Remove supernatant, collect remaining ethanol by quick centrifugation and pipette all remaining liquid using a 10 μl pipette. 
19. Resuspend each pellet in 6 ul H2O (DEPC-treated or RNase free).
SECTION: 3&#x27; Ligation
20. 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. 
SECTION: 3&#x27; Ligation
21. Incubate RNA + srBC 3’ 5 min at 70˚C, snap-cool on ice.
SECTION: 3&#x27; Ligation
22.        Set up master mix at room temperature:
 

SECTION: 3&#x27; Ligation
23. 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.
SECTION: Size selection (PAGE)
24. 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.
SECTION: Size selection (PAGE)
25. 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.
SECTION: Size selection (PAGE)
26. 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.
SECTION: Size selection (PAGE)
27. 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. 
SECTION: Size selection (PAGE)
28. 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).
SECTION: Size selection (PAGE)
29. 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.
SECTION: Size selection (PAGE)
30. 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. 
SECTION: Size selection (PAGE)
31. 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.
SECTION: Size selection (PAGE)
32. Add 800 µl 0.3 M NaCl + 0.1% SDS and allow RNA to elute overnight at room temperature, while rotating.
SECTION: EtOh precipitation
33. 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.
SECTION: 5’ linker ligation
36. Incubate RNA + adapter mix 5 min at 70˚C, snap-cool on ice.
SECTION: Total RNA isolation
9.4. Air dry pellet for approx. 5 min or until it becomes translucent.
18.4. Air dry pellet for approx. 5 min or until it becomes translucent.
SECTION: 5’ linker ligation
37. Prepare ligation master mix (volumes are calculated for one sample):

 

SECTION: EtOh precipitation
35. Without air-drying the pellet, dissolve small RNAs in 6 µl H2O + 1 µl 5’ adapter (10 µM).
SECTION: EtOh precipitation
34. 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.
SECTION: Purification using RNA Clean &amp; Concentrator 5 (Zymo)
40. Add 80 µl H2O and mix by pipetting.
SECTION: 5’ linker ligation
38. Add to each sample 13 µl of master mix, mix and give it a quick spin.
SECTION: 5’ linker ligation
39. Incubate o/n at 4˚C.

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

Note – pre-heat PCR block to 50˚C.
SECTION: Reverse transcription
44. RT reaction master mix:
 

SECTION: Reverse transcription
45. 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.
SECTION: Reverse transcription
46. Add 8 µl ExoSAP-IT and incubate 15 min at 37˚C followed by heat inactivation at 80˚C for 15 min.
SECTION: Reverse transcription
47. On ice, add 2 µl H2O for a total volume of 30 µl.
SECTION: qPCR amplification
48. 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. 
SECTION: qPCR amplification
49. Prepare also in duplicates 50 µl of fluorescent standard (STD) 3 and 4.
SECTION: qPCR amplification
50. Close strip with an optical flat-cap, vortex, and spin down.

SECTION: qPCR amplification
51. 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.

 


 


SECTION: Gel purification of amplified cDNA
52. 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.
SECTION: Gel purification of amplified cDNA
53. 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)
SECTION: Gel purification of amplified cDNA
54. 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.
SECTION: Gel purification of amplified cDNA
55. 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.


 

SECTION: Gel purification of amplified cDNA
56. 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.
SECTION: Gel purification of amplified cDNA
57. 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.
SECTION: Gel purification of amplified cDNA
58. Add 200 µl of DNA Wash Buffer to the column, centrifuge 1 minute and discard the flow-through; repeat the wash step once more.
SECTION: Gel purification of amplified cDNA
59. 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.
SECTION: Bioinformatics and data analysis
60. 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. 
SECTION: Bioinformatics and data analysis
61. Custom NextFlow pipelines for processing can be found on GitHub under https://github.com/popitsch/pysrna. In short:

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