title: GSM8170662: A549 cells, YTHDC1 CLIP-seq rep2; Homo sapiens; RIP-Seq
library_construction_protocol: 30x106 A549 per condition were crosslinked with 1% formaldehyde in 1X PBS for 10 minutes in agitation. Crosslinking was quenched with final concentration of 125 mM Glycine (Bio-Rad). Cells were fractionated as described by Neugebauer's lab (Brugiolo et al., 2017). After obtaining chromatin extracts, pellet was sheared by sonication in a Bioruptor device for 5 cycles (30''ON-30''OFF). We cleared nuclear extract by centrifugating 30 minutes at max speed and incubation with 50 µL Protein A/G Dynabeads for 1 hour at 4ºC. Chromatin extract was incubated with primary antibodies (Table S4) overnight at 4ºC in rotation. The following day, 50 µL Protein A/G Dynabeads where washed and mixed to antibody-hybridized nuclear extract for 6 hours at 4ºC. Beads were washed following the next order: 1 wash of Low-Salt Buffer (150 mM NaCl, 20 mM Tris [pH 8], 2 mM EDTA, 0.1% SDS, 1% Triton x-100), 1 wash of High-Buffer (500 mM NaCl, 20 mM Tris [pH 8], 2 mM EDTA, 0.1% SDS, 1% Triton x-100), 1 wash of LiCl Buffer (0.25 M LiCl, 1% IGEPAL, 1% Na-Deoxycholate [Na-DOC], 1 mM EDTA, 10 mM Tris [pH 8]) and 2 washes with TE Buffer (10 mM Tris, 1 mM EDTA [pH 8]). Beads are resuspended in 250 µL of Elution Buffer (25 mM Tris [pH 7.5], 5 mM EDTA, 0.5% SDS) supplemented with 10 µL of 5 M NaCl, 10 µL 1M Tris pH 7, 5 uL 0.5 M EDTA and 20 µg Proteinase K for 4 hours at 65ºC to promote proper decrosslinking of chromatin from immunoprecipitated proteins. RNA was isolated with TRI Reagent (Sigma-Aldrich) isolation and resuspended in 50 µL of water. Samples were treated with TURBO DNAse I (Invitrogen) for 45 minutes at 37ºC. After that time, a second RNA extraction with TRI Reagent was performed to removed TURBO DNAse I and final quantification by Qubit4 fluorometer (Invitrogen). SMARTer® Stranded Total RNA-Seq Kit v3