title: ChIP-Seq of Mus Musculus: young male/female spleen
design: Nave CD8+ T cells from Kmt2d WT and Kmt2d KO mice were sorted by FACS and stimulated with aCD3/CD28 in vitro for 24 hours. Human CD4 nave cells were sorted for spike in control. Cells were digested in digestion buffer (50mM Tris-HCl, 1mM CaCl2, 0.2% Triton X-100, 5 mM sodium butylate with proteinase inhibitor cocktail and 0.5 mM PMSF). Digested human CD4N cells were spiked into mouse cell digestions at the ratio of 10%. 0.2U MNase (ThermoFisher) was added per million cells and incubated at 37C for 40 minutes and sonicated (Diagenode) with a power setting of 5 for 4 repeats of 30 seconds followed by 30 seconds break between each repeat in ice-cold water to obtain the average size between 100-300 bps. The sonicated products were dialyzed with 10000 MWCO Cassette G2 (ThermoFisher) in RIPA buffer for 2 hours at 4C, and then incubated with Dynabeads Protein G (ThermoFisher) coated with 4 mg of anti-H3K4me1 antibody or non-specific IgG (Millipore) for overnight. The precipitated beads were washed with RIPA, LiCl and TE buffer sequentially, and recovered overnight at 65C with TE supplemented with 10% SDS and proteinase K (MBiotech). The ChIP DNA was purified with phenol/chloroform (Invitrogen) and precipitated. For sequencing, adapters were ligated to the precipitated DNA fragments or the input DNA to construct a sequencing library according to the manufacturers protocol (Illumina). In short, the fragments were end repaired using DNA polymerases and kinases. The fragments were A tailed on the 3 end and then T tailed illumine sequencing adapters, including unique indexes for each sample, were ligated to both ends of the DNA fragments.  These library samples were then size selected (~250-400 bases) on a 4.5% agarose gel. An 18-cycle PCR amplification was performed to enrich for fragments containing adaptors on both ends.  These amplified libraries were then tested for concentration and equal molar amounts of each were pooled and used to generate clusters onboard the Illumina HiSeq 2500 sequencer.  These clusters were then sequenced, with Illumina HiSeq Rapid Run Reagents, for 68 cycles then for an additional 7 cycles to determine the indexs present in each unique cluster. The images generated were used to generate sequence reads:  Using both lanes of the Illumina Rapid run flowcell we obtained ~500 million reads with 445 million reads passing the Illumina quality filter to be used for generating the actual sequencing reads.  The index reads were between 10.2% and 14.8% for the 8 samples with an expected average of 12.5% for each.  Of the passed filter reads, >94.5% had a quality score of Q30.