GSM7426458: ChIPseq_CH12_WT_Med23_r1; Mus musculus; ChIP-Seq
Gro-seq:  Nuclei was extracted from 8-12 million cells grown on 10 cm plates and after run-on reaction the  RNA was extracted with Trizol LS Reagent (Invitrogen, Carlsbad, CA, USA). RNA was treated. ChIP-Seq: Cultured cells were fixed with 1% formaldehyde (Sigma) for 10' at 37°C. Fixation was quenched by addition of glycine (Sigma) at a final concentration of 125mM. Twenty million fixed cells were washed with PBS and resuspended in 1ml of RIPA buffer (10mM Tris [pH 7.6], 1mM EDTA, 0.1% SDS, 0.1% sodium deoxycholate, 1% Triton X-100, 1× Complete Mini EDTA free proteinase inhibitor (Roche)) or stored at−80°C until further processing. Sonication was performed using Covaris S2 sonicator at duty cycle 20%, intensity 5, cycle/burst 200 for 30min or Branson sonifier at amplitude 35%, 12 cycles of 20” sonication and 30” of pause.   with TURBO DNase (Ambion), fragmented using RNA Fragmentation Reagents (Ambion) and  purified by running through P-30 column (Bio-Rad, Hercules, CA, USA). Fragmented RNA was  dephosphorylated with PNK (New England Biolabs, Ipswich, MA, USA) followed by heatinactivation.  Dephosphorylation reactions were purified using anti-BrdU beads (SantaCruz  Biotech, Santa Cruz, CA, USA) and precipitated overnight. Poly(A)-tailing and cDNA synthesis  was performed the next day as described in (Wang et al., 2011). However, for reverse  transcription oligos with custom barcodes (underlined) were used : 5'-Phos CA/TG/AC/GT  GATCGTCGGACTGTAGAACTCT /idSp/CAAGCAGAAGACGGCATACGA  TTTTTTTTTTTTTTTTTTTTVN-3'. After cDNA synthesis, Exonuclease I (New England  Biolabs; 30 min) was used to catalyze the removal of excess oligo. Enzyme was inactivated and  RNA hydrolyzed by alkaline treatment (100 mM NaOH) and heat (25 min, 95°C). The cDNA  fragments of were purified on a denaturing Novex 10% polyacrylamide TBE-urea gel  (Invitrogen). The recovered cDNA was circularized, linearized, amplified for 10-14 cycles. The  final product was ran on Novex 10%TBE gel, gel purified and cleaned-up using ChIP DNA  clean & Concentrator Kit (Zymo Research Corporation, Irvine, CA, USA).   mRNA-seq: Total  RNA  from  CH12  cells  was  isolated  by  Trizol  extraction. mRNA   was   then   isolated . ERCC-spike-in was added on some samples for quantative comparison between two conditions. ChIP-Seq :  Library was prepared in Ovation SP Ultralow library system (Nugen). 50 cycles of sequencing data were acquired on the HiSeq 3000 or Novaseq 6000 (Illumina).   mRNA-seq: standard RNA-Seq   library   preparation   was performed following Illuminaǯs RNA-Seq protocol v2. 50 cycles of sequencing data were acquired on Novaseq 6000 (Illumina).  Gro-seq: 50 cycles of sequencing data were acquired on the HiSeq 2500 (Illumina).