GSM8266774: Q45Q19, IP_2; Homo sapiens; RIP-Seq
Cells were lysed in cold CLIP lysis buffer (50mM Tris-HCl pH 7.4, 100mM NaCl, 1% NP-40, 0.1% SDS, 0.5% sodium deoxycholate, 1X protease inhibitor, and RNasin plus ribonuclease inhibitor). Followed by sonication in Bioruptor at “low” setting, 4°C for 5 min with 30 sec on and 30 sec off. 4 Units of DNase were added, and the tubes were incubated in a thermomixer at 37°C with shaking at 1200 rpm for 30 min. Next, the tubes were transferred on ice and immediately 50mM final concentration of EDTA was added. The tubes were centrifuged at 20,000xg at 4°C for 15 min to pellet debris. The supernatant was transferred to a new tube. The beads with EPR5526 (Abcam) antibody were rotated at 4°C for 1-2 hours and cell lysates were added to the prepared magnetic beads and the tubes were kept rotating at 4°C overnight. Next day, the tubes were placed in a magnetic rack and the cell lysate flowthrough was collected. The beads were washed 2x with cold high salt wash buffer (50mM Tris-HCl pH 7.4, 500mM NaCl, 1mM EDTA, 1% NP-40, 0.1% SDS, 0.5% sodium deoxycholate) and 1x with cold CLIP lysis buffer. 150uL of Proteinase K buffer (50mM Tris-HCl pH 7.4, 150mM NaCl, 1mM MgCl2, 0.05% NP-40, 1% sodium deoxycholate, and 1.2mg/mL Proteinase K) was added to the beads and incubated at 55°C for 30 min while shaking to digest proteins. After this reverse cross-linking step, the tubes were gently centrifuged using a table-top centrifuge and placed on a magnetic rack. The supernatants were transferred to new tubes and 600µL TRIzol reagent was added, and the tubes were incubated for 10 min at room temperature. The RNA was further purified using Direct-Zol RNA miniprep plus kit (Zymo Research, Cat. no. R2071). The purified RNA was eluted in RNases and DNase free water, and concentrations were estimated by nanodrop prior to library preparation. RIP-Seq libraries were prepared by using SMARTer Stranded Total RNA-Seq Kit version 2 (Pico Input Mammalian; Takara, 634413) as per the manufacturer's instructions. Subsequently, the libraries were subjected to paired-end sequencing with a read length of 150 bp on the Illumina HiSeq X Ten platform, ensuring a minimum sequencing depth of 30 million reads per sample.