GSM7523091: dAT1_CDM_HA_Brg1_rep2; Mus musculus; RIP-Seq
Mouse ESCs were fixed in 0.3% methanol-free formaldehyde for 30 min at 4 °C. Formaldehyde was quenched with 125 mM glycine for 5 min at room temperature (RT). Cells were washed three times in PBS at RT and resuspended at a final concentration of 10 million cells/mL. Cells were resuspended in 0.5 ml radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris-HCl pH 8, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 5 mM EDTA, 150 mM KCl) plus 0.5 mM DTT with 1× protease inhibitors (Sigma) and 2.5 μl RNAsin, and incubated on ice for 10 min prior to lysing using a Bioruptor (Diagenode) for two cycles of 30 s on and 1 min off, followed by centrifugation at 4 °C for 10 min at maximum speed. Protein A magnetic beads (Biorad) were pre-conjugated with antibody for 2 hr at 4 °C. Cells were then incubated overnight at 4 °C with antibody-conjugated beads. Beads were washed consecutively with fRIP buffer (25 mM Trix-HCl pH 7.5, 5 mM EDTA, 0.5% Ipegal CA-630, 150 mM KCl), three times in ChIP buffer (50 mM Tris-HCl pH 7.5, 140 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS), 1× in high salt buffer (ChIP buffer, but with 500 mM NaCl) and 1× in fRIP buffer. All washes were performed for 5 min at 4 °C. After the final wash, beads were resuspended in 100 μl of 1× reverse crosslinking buffer (3× PBS, 6% N-lauroyl sarcosine, 30 mM EDTA) supplemented with 5 mM DTT, 20 μl proteinase K and 1 μl SUPERaseIN and incubated 1 h at 42 °C, 1 h at 55 °C, and 30 min at 65 °C. Following elution and crosslink reversal, Trizol was used to extract RNA. Finally, RNA in the aqueous phase was supplemented with 1 volume of ethanol and purified using a Zymo-spin IC column, including the on-column DNase digestion, as per the manufacturer's instruction. RNA was eluted in 15 μl double distilled water (ddH20). RNA-seq libraries were prepared by using equal volume of the immunoprecipitate and included 1 μl of 1:250 μl dilution of ERCC spike-in mix 1 (Life Technologies). Libraries were then prepared using the Kapa Ribo-zero kit following the manufacturer's instructions, pooled, and sequenced using single-end 75-bp reads on an Illumina Nextseq 500.