GSM7946721: DMS seq WT D0 rep2; Mus musculus; OTHER
Frozen cells pellets are placed on dry ice, resuspended in nuclei isolation buffer (15 mM Tris-HCl pH 7.5, 60 mM KCl, 15 mM NaCl, 5 mM MgCl2, 1 mM CaCl2, 250 mM Sucrose, 0.3 % Igepal CA-630, 0.1% βME and 10 mM ribonucleoside vanadyl complex). The solution was repeatedly pipetted until the frozen pellet is completely dissolved. Nuclei were collected at 600 g and 4 °C for 5 min and washed twice with the nuclei isolation buffer without Igepal CA-630. To remove soluble nuclear fraction, nuclei pellet was resuspended in 500 ul NUN1 buffer (20 mM Tri-HCl, pH 8.0, 75mM NaCl, 0.5 mM EDTA, 50% Glycerol and proteinase inhibitor 1xComplete followed by 4.8 ml NUN2 buffer (20 mM HEPES-KOH pH 7.6, 7.5 mM MgCl2, 0.2 mM EDTA, 300 mM NaCl, 1 M Urea, 1% Igepal CA-630). The solution was incubated on ice for 15 min with brief vortexing every 5 mins. An insoluble chromatin pellet was obtained by centrifugation at 10000 g at 4 °C for 15 min centrifugation. 300 ml Trizol was added to the chromatin pellets, and under freeze-thaw cycles three times.  Then the pellets were ground with eppendorf tube pestles until completely resuspended in trizol. Additional 700 ml Trizol was applied on the top of the ground mixture and the remaining RNA isolation procedure was performed as following standard Trizol protocol. Poly A+ RNA was collected from 100 - 250 ug chromatin-bound RNA using Oligotex Direct mRNA kit. 1 - 5 ug of poly A+ RNA was chemically fragmented at 95 °C for 8 min in RNA fragmentation buffer (100 mM Tris-HCl pH 8 and 2 mM MgCl2). The fragmented RNA was ethanol precipitated, resuspended in urea gel loading buffer (1x TBE, 3.5M Urea, 0.005% Bromophenol blue, and 0.025% Xylene), and subjected to denaturing urea 8% acrylamide gel fraction. 60- 80 nt RNA fragment was excised and recovered from the gel. RNA samples are dephosphorylated with polynucleotide kinase (PNK) (NEB M0201S) in 10 ml solution comprising 1 ml 10x T4 RNA ligase buffer (NEB, B0216), 1.5 ml T4 PNK and 0.5 ml SUPERase•In RNase Inhibitor (Invitrogen, AM2696) at 37 °C for 5 hours. The samples are ligated to the 3' adaptor by directly adding 6 ml 50 % PEG8000, 1 ml 10x T4 RNA ligase buffer, 2 ml 20 mM adenylated 3 DNA adaptor, and 1 ml T4 RNA Ligase 2 truncated K227Q (NEB, M0373) to the dephosphorylated mixture above and incubating 16 °C for 16 hours. Samples are ethanol precipitated and gel fractionated again, adaptor-ligated products appearing at 85 -105 nt position were gel-purified and resuspended in 13 ul water. For reverse transcription, 13 ml adaptor-ligated RNA was mixed with 4 ml 5x RT buffer (100 mM Tris-HCl pH 7.5, 750 mM KCl (or LiCl), 15 mM MgCl2), 1 ml 10 mM dNTP mix, 2 ml 10 mM phosphorylated iSp18 PE Reverse oligo and 0.1 ul SUPERase•In RNase Inhibitor. The mixture was incubated in PCR machine at 80 °C for 2 min and then 42 °C for 5min, 1 ml 0.1M DTT and 1 ml SuperScript III reverse transcriptase (Invitrogen, 18080085) was added to the mixture while PCR tubes are in the thermocycling block. After incubation at 42 °C for 10 min for cDNA synthesis, samples are moved on ice and then 2 ml 1 M NaOH was added to the reaction mixture. To hydrolyze RNA, samples were treated at 98°C for 15 minutes, and 2 ml 1 M HCl was applied to neutralize the solution. Resulting cDNAs were ethanol precipitated and urea gel fractionated, smearing band appearing at the 78 -105 nt length was purified from the gel. cDNA samples are circularized using 50 U CircLigase (Epicentre, CL4111K) at 60°C for 12 hours. Circularized cDNA was amplified using RT stop amp primer (Supplementary Table 7) and KAPA HiFi PCR master mix (Roche, KK2601) for 8–11 cycles of PCR. Second size selection was performed with native TBE- 8% acrylamide gel fractionation, PCR bands at 78- 105 bp are purified from the gel. 4 - 5 cycles of additional PCR reactions were run with NEBnext multiplex oligos (NEB, E7335) to introduce Illumina library sequences to the RT-stop profile library. ZR small-RNA PAGE Recovery Kit (Zymo Research, R1070) and SYBR Gold (Invitrogen, S11494) staining was used for gel purification during the preparation process.