title: GSM4785375: HNP1 cell line YTHDF2 iCLIP rep1; Homo sapiens; RIP-Seq
library_construction_protocol: Crosslinked cell pellets were resuspended in 100 µL of cold 1% SDS + 10mM DTT + 10X Halt protease inhibitor cocktail (Thermo Fisher) solution to dissociate YTHDF2 complexes, and sonicated using a Branson Digital Sonifier Model 450 fitted with 3.125 mm tapered micro tip probe on ice at 10% amplitude (2s ON, 10s OFF cycle, total 20s). The pellet was topped up to 1mL of 1X RIPA buffer (50mM Tris-HCl Ph 7.5, 200mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS), and sonicated again at 10% amplitude (2s ON, 10s OFF cycle, total 20s) YTHDF2 iCLIP libraries were prepared as previously described (PMID: 27602518) with the following modifications: DNase I (New England Biolabs) and RNase I (Thermo Fisher) was used to digest DNA and RNA in the cell pellet (high RNase I dilution = 1:5, low RNase I dilution = 1:50 or 1:100 depending on cell line). 4 ug of YTHDF2 antibody (Aviva Systems Biology, ARP67917) was used for immunoprecipitation of each 1mg of cell lysate. Equivalent amounts of normal rabbit IgG (Cell Signalling Technology) were used for immunoprecipitation as a negative control. YTHDF2 antibodies or Rabbit IgG were first bound to Pierce Protein A/G Magnetic Beads (Thermo Fisher) in RIPA buffer for 1h at 4 °C. Immunoprecipitation was performed in RIPA buffer at 4 °C overnight. RNA bound to YTHDF2 was ligated to adaptors and purified. SuperScript IV and primers with unique barcodes were used for reverse transcription to tag each replicate. The medium cDNA fraction (80 – 100nt) was extracted by gel purification, and sequenced on the Illumina HiSeq4000 (paired end, 150 bases).