GSM2587411: aB24h_wt_input_rep1; Mus musculus; ChIP-Seq
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 in situ HiC libraries at Rao et al., (Cell, 2014 Dec; 159:1-16)                                                                                                                                                                                          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).                                                                                  4C-seq:  The  4C  assay  was  performed  as  previously  described  van  de  Werken  et  al.  (2012) with  minor  modifications.  Ten  million  of  CH12  B  cell  line  were  crosslinked  in  2% formaldehyde  at  37°C  for  10 min.  The  reaction  was  quenched  by  the  addition  of glycine (final concentration of 0.125 M). Cells were then washed with cold PBS and lysed  (10 mM  Tris-HCl,  pH  8.0,  10 mM  NaCl,  0.2%  NP-40, 1×  complete  protease inhibitors  [Roche])  at  4°C  for  1 hr.  Nuclei  were  incubated  at  65°C  for  30 min,  37°C for  30 min  in  500 μl  of  restriction  buffer  (New  England  BioLabs  DpnII  buffer) containing  0.3%  SDS.  To  sequester  SDS,  Triton  X-100  was  then  added  to  a  final concentration  of  1.8%.  DNA  digestion  was  performed  with  400 U of  DpnII  (New England  Biolabs)  at  37°C  overnight.  After  heat  inactivation  (65°C for  30 min),  the reaction was diluted to a final volume of 7 ml with ligation buffer containing 100 U T4 DNA Ligase (Roche) and incubated at 16°C overnight. Samples were then treated with  500 μg  Proteinase  K  (Ambion)  and  incubated  overnight  at  65°C  to  reverse formaldehyde crosslinking. DNA was then purified by phenol extraction and ethanol precipitation.  For  circularization,  the  ligation  junctions  were  digested  with  Csp6I (Fermentas) at 37°C overnight. After enzyme inactivation and phenol extraction, the DNA   was   religated   in   a   7 ml   volume   (1,000 U   T4   DNA   Ligase,   Roche).   Three micrograms  of  4C  library  DNA  was  amplified  with  Expand  Long  template  PCR System  (Roche).  Thermal  cycle  conditions  were  DNA  denaturing  for  2 min  at  94°C, followed by 30 cycles of 15 s at 94°C, 1 min at 58°C, 3 min at 68°C, and a final step of 7 min  at  68°C.    Bait  was  amplified  with  inverse  PCR  primers  as  follows: 3RR with DpnII:         _4C         5′-         GCTTATCTGTAAAGAATGGGTC-3′,         3RR_Csp6i         5′-GGCCTTAGAAGGCTCTGTAC-3′.  4C-amplified  DNA  was  microsequenced  with  the Illumina platform.                mRNA-seq: Total  RNA  from  106 of  WT  or  ZF9-11  CH12  cells  was  isolated  by  Trizol  extraction.mRNA   was   then   isolated .                                                                                                                                                                                             local Hi-C: 500ng of Hi-C library (8 cycles of amplification) was mixed with 2.5 µg of mouse Cot-1 DNA and 10µg of salomon sperm DNA in a total volume of 25µl. The sample was heated to 95℃ for 5 minutes. Next the temperature was diminished to 65℃ and the samples incubated for at least 5 minutes at 65℃. In the meantime 6µl of a RNA probe mixture (500ng of RNA probes, 20U of supreRNAseIn) and 33µl of hybridization buffer (10xSSPE, 10xDenhardt buffer, 10mM EDTA, 0.2% SDS) were incubated at  65℃. Next, the hybridization buffer and the RNA probe mixture were added to the sample. The final mix was incubated at 65℃ for 24 hours. Following the hybridization, 50 µl of MyOne Streptavidin T1 beads were washed 3 times with Bind-and-Wash buffer (1M NaCl, 10 mM Tris-HCl, pH=7.5, 1mM EDTA) and then resuspended in 134 µl of Bind-and-Wash buffer and added to the sample. The mixture was incubated for 30 minutes at room temperature with occasional swirling. Next, beads were separated using a magnet and the supernatant discarded. Beads were then washed once with 200µl of low-stringency buffer (1xSSC, 0.1%SDS) and incubated for 15 minutes at RT. Beads were separated and washed 3 times with high strin