title: GSM2652807: aB_myc_nih_rep3; Mus musculus; OTHER
library_construction_protocol: 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 125 mM. Twenty million fixed cells were washed with PBS and resuspended in 1 ml of RIPA buffer (10 mM Tris [pH 7.6], 1 mM 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 30 min or Branson sonifier at amplitude 35%, 12 cycles of 20” sonication and 30” of pause. For native chip, chromatin was digested with Mnase (Sigma) in digestion buffer (50 mM Tris-HCl, pH7.6, 1 mM CaCl2, 0.2% Triton X-100, butyrate 5 mM) for 5’ at  37°C and dialyzed against RIPA buffer for 2hrs at 4°C. Five micrograms of antibody were incubated with 40 μl of Dynabeads Protein A (or G) for 40 min at room temperature. Antibody-bound beads were added to 500 μl of sonicated or Mnase-digested chromatin, incubated at 4°C overnight, and washed twice with RIPA buffer, twice with RIPA buffer containing 0.3M NaCl, twice with LiCl buffer (0.25 M LiCl, 0.5% Igepal-630, 0.5% sodium deoxycholate), once with TE (pH 8.0) plus 0.2% Triton X-100, and once with TE (pH 8.0). Crosslinking was reversed by incubating the beads at 65°C for 4 hr in the presence of 0.3% SDS and 1 mg/ml Proteinase K. ChIP DNA was purified by phenol-chloroform extraction followed by ethanol precipitation.  MNase-Seq: 4x107 resting and activated B cells were digested with 24 different concentrations of MnaseI. After an initial RT-qPCR analysis and curve-fitting, the MnaseI concentrations for the most representative nucleosomal fraction was determined. The lowest three nucleosomal fractions (which corresponded to MnaseI concentrations of 0U, 0U, and 0.0026U) were pooled together and used as input control. Nucleosomal fractions 16-18 (which corresponded to enzyme concentrations of 0.068U, 0.045U, and 0.03U) were also pooled for further processing.  Input samples were sonicated to obtain similar fragment sizes as the MnaseI-digested samples. DNA was then concentrated and each sample was spiked with 0.055mg of l DNA (also sonicated to match the sample size as was input DNA).                                                                                                                                                                                             HiC-Seq:  we used the same protocol for situ HiC libraries at Rao et al., (Cell, 2014 Dec; 159:1-16)                                                                                                                                                                         ATAC-Seq: we followed the protocol at Buenrostri J.D. et al. (Nat Methods, 2013 Dec; 10(12):1213-8)                 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  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). ChIP-Seq :  Library was prepared in Ovation SP Ultralow library system (Nugen). 50 cycles of sequencing data were acquired on the HiSeq 2000 or 2500 (Illumina).                                                                                                                                                      MNase-Seq: DNA was isolated and prepared for paired-end Illumina sequencing following the genomic DNA protocol. 50 cycles of sequencing data were acquired on the HiSeq 2000 or 2500 (Illumina)                                                        ATAC-Seq: DNA was isolated and prepared for paired-end Illumina sequencing following the genomic DNA protocol. 50 cycles of sequencing data were acquired on the HiSeq 2000 or 2500 (Illumina).                                   HiC-seq: DNA was isolated and prepared for paired-end Illumina sequencing following the genomic DNA protocol. 100 cycles of sequencing data were acquired on the HiSeq 2000 or 2500 (Illumina).                                    Gro-seq: 50 cycles of sequencing data were acquired on the HiSeq 2000 (Illumina).
attributes.tag: GEO Accession
attributes.value: GSM2652807