GSM4567042: Dhx36_CLIP_Bio_Rep1; Mus musculus; OTHER
The cell pellet was resuspended in 1 ml of wash buffer (1xPBS, 0.1% SDS, 0.5% NP-40, 0.5% deoxycholate), mixed briefly and incubated on ice for 15 min. After sonicated twice for 10 sec each at the maximum setting (BRANSON, SONIFER cell disruptor 185) (output setting 4; timer Hold), cells were spun down at 13,000 rpm for 15 min at 4 °C. Then added the beads which coupled antibody to the cell lysate and incubate 4h at 4 °C. After immunoprecipitation, washed beads with wash buffer, high salt wash buffer (5xPBS, 0.1% SDS, 0.5% NP-40, 0.5% deoxycholate), and 1 × PNK buffer (50 mM Tris-HCl pH7.4, 10mM MgCl2, 0.5% NP-40). And then the beads was briefly incubated with 200 μl of 1 × MN buffer (50 mM Tris-HCl pH 8.0, 5 mM CaCl2) plus 0.12 U micrococcal nuclease for 10 min at 37 °C. The reaction was stopped by washing twice with 1 × PNK + EGTA buffer (50 mM Tris- HCl pH7.4, 20 mM EGTA, 0.5% NP-40) and twice with 1 × PNK buffer. After the fragmentation, beads were treated with Fast alkaline phosphatase for 15 min at 37 °C. Then stopped the reaction by washing beads twice with 1 × PNK + EGTA buffer and 1 × PNK buffer. The target RNAs in bead then were ligated the 3' linker by the T4 RNA Ligase 2, truncated KQ. After the ligation, protein and the binding RNA were elute by 1xLDS sample buffer for 10min at 70 °C. And run the 4-12% bis-tris gel and transfer membrane. After recover the membrane, proteinase K treatment was carried out to release the RNAs, and the RNAs were extracted with acid Phenol: chloroform and precipitated at -20°C ethanol. RNAs were pelleted at 13,000 rpm and washed twice with 75% ETOH before resuspending in 6.5 μl of nuclease-free water. The 5' linker were ligated by T4 RNA ligase for 4h at 16°C. And the RNAs were extracted like above, and washed twice with 75% ETOH before resuspending in 8 μl of nuclease-free water. Reverse transcription was performed by addition of 1 μl 10 mM dNTPs and 1 μl 10 mM RT primer, incubated at 65 °C for 5 min. And then added the 10 μl of RT mix (2 μl of 10 × Superscript RT buffer, 1 μl of 100 mM DTT, 1 μl of RNase inhibitor, 1 μl of Superscript III reverse transcriptase) to the mixture and incubation at 50 °C for 60 min, and then 80 °C for 5 min. The 1st round PCR was performed with 1 μl of 10 μM AD primer, 1 μl of 10 μM PB primer, 1 μl 10 mM dNTPs, 10 μl of 5 × phusion HF buffer, 0.5 μl of phusion taq polymerase. The 70 bp to 110 bp of PCR products were purified from TBE gel. Then the 2nd round PCR was performed with 0.5 μl of 10 μM index primer, 0.5 μl of 10 μM PB primer, 0.4 μl 10 mM dNTPs, 4 μl of 5 × phusion HF buffer, 0.15 μl of phusion taq polymerase and purified the 110 bp to 170 bp of PCR products. The DNA concentration was measured by Qubit and sequenced by Illumina single-end sequencing.
GEO Accession
GSM4567042