title: GSM1185223: H3K4me3 ChIP-seq (Rag1 -/- DN thymocytes); Mus musculus; ChIP-Seq
library_construction_protocol: DN cells were crosslinked with 1% formaldehyde for 10 minutes at room temperature. Subsequently, the lysates were sonicated using a Diagenode Biorupter. The chromatin solution was precleared with salmon sperm DNA-protein A agarose beads. The lysate was immunoprecipitated using the following antibodies: H3ac was purchased from EMD Millipore (Billerica, MA), H3K4me3 from Active Motif (Carlsbad, CA). Immune complexes were isolated with protein A agarose beads.  Following elution, chromatin-antibody complexes and input DNA were reverse crosslinked by heating at 65°C overnight.  The DNA was purified using the Qiagen DNA purification kit. The sequencing libraries were prepared using 10 ng of DNA. ChIP DNA sample ends were repaired using the recommended Illumina protocol, including T4 DNA polymerase, Klenow Large Fragment, and T4 polynucleotide kinase. DNA products were purified using the DNA Clean&Concentrator- 5 Kit (Zymo Research). The DNA ends were A-tailed with Klenow Large Fragment (3′→5′ exo-) at 37 °C for 30 min. DNA products were again purified using the DNA Clean&Concentrator- 5 Kit. Next, Illumina Paired End-adapter oligonucleotides (2), at a concentration of 0.33 μM, were ligated to the A-tailed cDNA ends with T4 DNA ligase. DNA products were purified using the DNA Clean&Concentrator-5 Kit. The DNA library products were separated on a 2% (wt/vol) agarose gel, and products corresponding to a size of ∼200–250 bases were removed from the gel and cleaned using the Agencourt SPRI system (Beckman). The DNA material was PCR-amplified with Phusion Polymerase (Finnzymes) with 0.6 μM PCR primers PE 1.0 and PE 2.0 (Illumina) for 15 cycles. The amplified DNA products were further purified on 2% (wt/vol) agarose gel, excised, and isolated again using the ZymocleanGel DNA recovery kit (Zymo Research). The purified DNA library was quantitated using the Qubit quantitation platform (Invitrogen) and sized using the 2100 Bioanalyzer (Agilent). DNA products were then denatured in 0.1 N of NaOH and diluted to a final concentration of 10 pM before being loaded onto the Illumina single read flow cell for 100 base sequencing by synthesis on the Illumina HiSeq2000.