GSM1356574: U2OS_+Dox_pLKO-empty_replicate2_RNAseq; Homo sapiens; RNA-Seq
For ChIP-seq cells were crosslinked with 1 % formaldehyde for 10' at 37°C. After cell lysis nuclei were resuspended in RIPA buffer and sonicated (Bransson) until average fragment size was below 500 bps. Antibodies were bound to Protein-A/G-sepharose or Dyna beads and incubated with the chromatin. After sequential washing and elution with 1 % SDS, crosslinking was reverted and DNA was purified using Qiagen PCR purification columns. Libraries were contructed following manufactor's intructions (NEBNext ChIP-Seq Sample Prep Kit). 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. Afterwards, DNA was enriched with 18 PCR cycles, fragment size was controlled with Biorad Experion system an quantified using picogreen assay. Libraries were sequenced on an Illumina Genome Analyzer IIx following the manufactor's instructions. For RNAseq, poly-A RNA was isolated from total with Sera-Mag Oligo(dT) Magnetic Particles (Thermo Scientific). Library preparation was performed by using the NEBnext® mRNA Library Prep Master Mix set for Illumina (E6100S/L) following the instruction manual. Briefly, poly-A RNA was frangmented to generate 200 nucleotides fragments. First and second strand synthesis was performed and the resulting cDNA was end-repaired, ligated to NEBnext Adaptor, size selected (250bps) and purified with Qiagen gel extraction kit. cDNA was then amplified with 12-15 cycle of PCR and the resulting library was subjected to Illumina GAIIx sequencing according to the manufacturer instructions.