GSM4145964: ChIPSeq_HUVEC_H3K27me3_Nox_rep1_set1; Homo sapiens; ChIP-Seq
ChIP-Seq: Formaldehyde (37% liquid stock solution) was added drop by drop to the culture medium on 10cm plates to a final concentration of 1% and incubated at room temperature for 10 min. Glycine was added to a final concentration of 125mM at room temperature for 5 min to stop cross-linking. The culture medium was removed, and cells were washed twice with ice-cold 0.1% BSA in PBS. Cells were scraped and collected in MNase buffer (10mM Tris pH 7.4, 10mM NaCl, 5mM MgCl2, 0.5% IGEPAL NP-40, 1x protease inhibitor cocktail PIC, Roche), 1 mM PMSF) and incubated on ice for 10 min. Cells were spun down, washed with 1mL MNase buffer, and spun down again. The supernatant was removed and the pellet was suspended to 300µL of SDS lysis buffer (0.5% SDS, 10mM EDTA, 50mM Tris-HCl pH 8.1, 1x PIC, 1x PMSF). Samples were sonicated usign Bioruptor for 26 cycles. RNA-Seq: RNA was purified using RNeasy Mini Kit and enriched for Poly(A)-RNA with MicroPoly(A) Purist Kit. ChIP-Seq: Lysates were clarified from sonicated nuclei and protein-DNA complexes were isolated with H3K27me3 Ab (Millipore #07-449). DNA was end-repaired using a combination of T4 DNA polymerase, E. coli DNA Pol I large fragment (Klenow polymerase) and T4 polynucleotide kinase. The blunt, phosphorylated ends were treated with Klenow fragment (minus exo) and dATP to yield a protruding 3- 'A' base for ligation of Illumina's adapters which have a single 'T' base overhang at the 3' end. After adapter ligation, ChIP DNA was PCR-amplified with Illumina Genomic adaptors or with NEXTflex DNA barcodes adaptors and library fragments were size-selected (150-250bp) from a 2% agarose gel. RNA-Seq: The RNA was base-hydrolyzed, dephosphorylated with PNK and purified using RNA Clean & Concentrator kit (Zymo). Poly(A)-tailing was followed by cDNA synthesis using complementary poly(T)-primers containing Illumina adapter sequences. Excess oligo was removed by Exonuclease I and cDNA fragments of were purified using ChIP DNA Clean & Concentrator kit. The recovered cDNA was RNaseH treated and circularized (CircLigase) and amplified for 11 cycles. The final product was ran on 10% TBE gel, gel purified (180-350 bp) and cleaned-up using ChIP DNA clean & Concentrator Kit.
GEO Accession
GSM4145964