title: GSM9077215: HCT116 cells, ZC3H4-3xFLAG, FLAG-IP; Homo sapiens; RIP-Seq
library_name: GSM9077215
library_construction_protocol: Crosslinked cells were lysed with iCLIP lysis buffer (50mM Tris-HCl pH 7.4, 100mM NaCl, 1% NP-40, 0.1% SDS, 0.5% Sodium deoxycholate, 1:200 Proteinase Inhibitor and sonicated, followed by 5 min treatment with RNase I  at 37°C to fragment RNA. RNA from IPs was dephosphorylated with FastAP for 15 min at 37°C. T4 PNK was added for 20 min at 37°C at low pH, and a color-balanced pair of 3′ RNA adaptors (RNA_X1A/X1B or RNA_X2A/X2B) was ligated with T4 RNA ligase for 75 min at 25°C. Input samples were dephosphorylated with FastAP and phosphorylated with T4 PNK for 20 min at 37°C at low pH, and the 3′ RNA adaptor, RiL19, was ligated with T4 RNA ligase for 75 min at 25°C. Reverse transcription was then performed with AffinityScript using the AR17 primer for 55 min at 45°C, followed by ExoSAP-IT treatment to remove unincorporated primers. RNA was then degraded by alkaline hydrolysis (1M NaOH, 70°C, 12 min), and the 3′ DNA adaptor, rand103Tr3, was ligated with T4 RNA ligase overnight at 25°C. The D5x/D7x pair of primers was used in a qPCR reaction to determine the required amplification cycles, followed by PCR with Q5 polymerase (NEB M0492) using Combinatorial dual (CD) index adapters and the libraries were purified using 1,8x solid-phase reversible immobilization (SPRI) beads (Agencourt AMPure XP, Beckman Coulter).