title: GSM9039571: IFE-1 CLIP Replicate 1 (CLIP); Caenorhabditis elegans; RIP-Seq
library_construction_protocol: Homogenized samples were treated with 250 U of RNase I (Ambion) at 37°C for 10 minutes with constant shaking at 1200 rpm prior to immunoprecipitation. Briefly, upon RNase I digestion, 2% of 5 mg worm lysate was taken as inputs and the rest of the lysate was incubated with 100 µl of anti-FLAG magnetic beads (Sigma) with a final concentration of 1:5 dilution of wash buffer (20 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 5 mM NaCl, 0.2% Tween-20) for 1 hour. IP samples were washed once with high-salt wash buffer (50 mM Tris-HCl (pH 7.5), 1 M NaCl, 1 mM EDTA, 0.1% SDS, 1% NP-40) and washed twice with the wash buffer. T4 PNK Minus reaction was performed on bead using T4 PNK Minus (NEB) at 37°C for 20 minutes with constant shaking at 1200 rpm, followed by 1x wash with high-salt wash buffer and 3x wash with wash buffer. IP samples were subjected to Fast AP (Thermo Scientific) reaction at 37°C for 10 minutes with constant shaking at 1200 rpm and PNK reaction with T4 PNK (NEB) at 37°C for 20 minutes with constant shaking at 1200 rpm without removing the FastAP reaction mixture. IP samples were washed 1x with high-salt wash buffer and 3x with wash buffer. 3' RNA adapter (CLASHInvRiL19) was ligated on bead using T4 RNA ligase (high-concentration, NEB) at RT for 75 minutes on a rotator. IP samples were washed 1x with wash buffer, 1x with high-salt wash buffer, and 2x with wash buffer. All input and IP samples were denatured using 4xNuPAGE™ sample buffer (Invitrogen) at 65°C for 10 minutes and resolved in NuPAGE™ 4-12% Bis-Tris gel (Invitrogen) at 150 V for 75 minutes, followed by transfer to nitrocellulose membrane at 30 V for overnight. After confirming the position of IFE-1 protein by western blot using FLAG antibody (Sigma), regions above IFE-1 protein (up to ~250 kDa) were excised for proteinase K (NEB) treatment at 37°C for 40 minutes with constant shaking at 1200 rpm. RNA was isolated from proteinase K treatment reaction using Zymo RNA clean & concentrator kit (Zymo Research). Upon RNA isolation, input samples were also treated with FastAP (Thermo Scientific) for 10 minutes and T4 PNK (NEB) for 20 minutes, both at 37°C with constant shaking at 1200 rpm, followed by clean up using Zymo RNA clean & concentrator kit (Zymo Research). 3' RNA adapter (CLASHInvRiL19) was ligated to input samples after incubation at 65°C for 2 minutes using T4 RNA ligase (high-concentration, NEB) at RT for 60 minutes with flicking every 15 minutes. Input samples were cleaned up using MyONE Silane beads (Invitrogen). Reverse transcription was performed for both IP and input samples after incubation at 65°C for 2 minutes using an RT primer (InvAR17) and then at 55°C for 20 minutes using Superscript IV (Invitrogen). The reaction was cleaned up using ExoSAP-IT (Applied Biosystems) and MyONE Silane beads (Invitrogen). Then, 5' DNA adapter (Invrand10_3Tr3) was ligated after incubation at 70°C for 2 minutes and then at RT for overnight using T4 RNA ligase (high-concentration, NEB) and 5' deadenylase (NEB). Samples were cleaned up using MyONE Silane beads (Invitrogen) and PCR amplified for 8 cycles (Input) and 13 cycles (IP) using Q5 High-Fidelity PCR master mix (NEB) and PCR forward and reverse primers with specific barcodes. Libraries were purified using Ampure XP beads (Beckman Coulter) and quantified using the D1000 high sensitivity screen tape system (Agilent, La Jolla, CA) before pooling. The pooled library was sequenced on NextSeq2000 (Illumina, San Diego, CA) at the center of bioinformatics and genomics (CGB) at Indiana University, Bloomington using paired-end sequencing.
identifiers.namespace: GEO
identifiers.id: GSM9039571_r1