GSM3358090: cll_20792t_h3k27me3_bam; Homo sapiens; ChIP-Seq
A minimum of 2 million purified human cells were used. Briefly, cells were fixed in a 1% methanol-free formaldehyde solution and then resuspended in sodium dodecyl sulfate (SDS) lysis buffer. Lysates were sonicated in an E220 focused-ultrasonicator (Covaris, Woburn, MA) to a desired fragment size distribution of 100–500 base pairs. Immunoprecipitation reactions were performed with the above-indicated antibodies, each on approximately 500,000 cells. ChIP assays were processed on an SX-8G IP-STAR Compact Automated System (Diagenode, Denville, NJ) using a direct ChIP protocol as previously described. Eluted chromatin fragments were then de-crosslinked and the DNA fragments purified using Agencourt AMPure XP beads (Beckman Coulter, Brea, CA). Barcoded immunoprecipitated DNA and input DNA were prepared using the NEBNext ChIP-seq Library Prep Master Mix Set for Illumina (#E6240, New England Biolabs, Ipswich, MA) and TruSeq Adaptors (Illumina) according to the manufacturer's protocol on an SX-8G IP-STAR Compact Automated System (Diagenode). Phusion High-Fidelity DNA Polymerase (New England Biolabs) and TruSeq PCR Primers (Illumina, San Diego, CA) were used to amplify the libraries, which were then purified to remove adaptor dimers using AMPure XP beads and multiplexed on the HiSeq 2000 (Illumina, San Diego, CA). dodecyl sulfate (SDS) lysis buffer. Lysates were sonicated in an E220 focused-ultrasonicator (Covaris, Woburn, MA) to a desired fragment size distribution of 100–500 base pairs. Immunoprecipitation reactions were performed with the above-indicated antibodies, each on approximately 500,000 cells. ChIP assays were processed on an SX-8G IP-STAR Compact Automated System (Diagenode, Denville, NJ) using a direct ChIP protocol as previously described45. Eluted chromatin fragments were then de-crosslinked and the DNA fragments purified using Agencourt AMPure XP beads (Beckman Coulter, Brea, CA). Barcoded immunoprecipitated DNA and input DNA were prepared using the NEBNext ChIP-seq Library Prep Master Mix Set for Illumina (#E6240, New England Biolabs, Ipswich, MA) and TruSeq Adaptors (Illumina) according to the manufacturer's protocol on an SX-8G IP-STAR Compact Automated System (Diagenode). Phusion High-Fidelity DNA Polymerase (New England Biolabs) and TruSeq PCR Primers (Illumina, San Diego, CA) were used to amplify the libraries, which were then purified to remove adaptor dimers using AMPure XP beads and multiplexed on the HiSeq 2000 (Illumina, San Diego, CA). RNA was extracted using Qiagen (Hilden, Germany) RNeasy columns according to the manufacturer's instructions. Subsequently, 500 ng of total RNA was used for polyA selection and TruSeq library preparation according to the instructions provided by Illumina (TruSeq RNA Sample Prep Kit v.2), with 8 cycles of PCR. Samples were barcoded and run on a HiSeq 4000 in a 125 bp paired-end mode, using the TruSeq SBS Kit v.3 (Illumina, San Diego, CA). An average of 75 million paired reads was generated per sample.
GEO
GSM3358090