title: GSM1430923: Ls174T_DMSO_IgG_ChIPseq; Homo sapiens; ChIP-Seq
library_construction_protocol: For ChIPseq cells were crosslinked with 1% formaldehyde at 37°C for 10min. Cells were lysed and after centrifugation nuclei were re-suspended in RIPA buffer. DNA was sonicated with a Branson sonifier to obtain DNA fragments ≤ 500 bp. Chromatin was pre-cleared with protein A- or protein G-sepharose beads (blocked with BSA) and immunoprecipitated with indicated antibodies. Chromatin/protein complexes were collected by incubation with Protein A- or Protein G-sepharose beads for 6 hrs. After several washings chromatin was eluted with 1 % SDS/0.1 M NaHCO3 and  crosslinking was reverted overnight. DNA was purified by Phenol/Chloroform extraction. For RNAseq total RNA was extracted using the RNeasy Mini Kit (Qiagen) including on-column DNase digestion. Poly-A RNA was isolated from total RNA using the NEBNext Poly(A)mRNA Magnetic Isolation Module (E7490). Libraries for ChIP-seq samples were contructed following manufactor's intructions using the NEBNext ChIP-Seq Library Prep Master Mix Set for Illumina (E6240). Briefly, ChIP DNA was end repaired,  A-tailed  and Illumina adaptors were ligated. DNA fragments of about 200 bps were cut out of a agarose gel and extracted with a Qiagen PCR purification column. Size-selected DNA was amplified with 18 PCR cycles. Libraries for the RNA-seq samples were constructed using the NEBNext Ultra RNA Library Prep Kit for Illumina (E7530) following the instruction manual. Briefly, poly-A RNA was frangmented to generate 200 nucleotides long fragments. First and second strand cDNS synthesis was performed and the resulting cDNA was end-repaired, ligated to NEBNext adaptors. The cDNA was size selected using Agencourt AMPure XP Beads (Beckman Coulter) and amplified with 13 cycles of PCR. For the resulting ChIP-seq and RNA-seq libraries, amplicon sizes and quantitiies were determined using the Biorad Experion system. Libraries were sequenced on an Illumina Genome Analyzer IIx following the manufactor's instructions.