GSM2731542: RNA-seq treatment replicate 3; Homo sapiens; RNA-Seq
Total RNA from 226LDM cells (three independent samples from the control and three from the treated population) was extracted using the TRIsure reagent (Bioline) according to the manufacturer’s guidelines. Briefly, cells grown in a T75 flask were washed twice with PBS, then scraped off and pelleted at 300 g for 5 min. Following incubation with TRIsure for 5 min at RT, chloroform was added and the sample was incubated for 15 min at RT. After centrifugation at 9,500 g for 15 min at 4oC, the top aqueous layer was carefully extracted and the genetic material was precipitated with isopropanol for 20 min on ice. After centrifugation (9.500 g / 15 min / 4oC) the pellet was washed twice in 75 % ethanol before air-drying the obtained RNA pellet. The RNA was solubilized in sterile water (40-50 μl) and heated for 10 min at 55oC. The pellet was stored at -80oC. ChIP was performed using the ZymoSpin kit (Zymo Research USA) following the manufacturer’s instructions. In brief, 5 x 106 of 226LDM cells from the control and the treated populations were cross-linked with formaldehyde. The cross-linking was quenched with glycine and the cells were washed twice with PBS with the addition of a protease inhibitor cocktail before pelleting at 1000 g for 1 min at 4oC. The pellet was lysed in Chromatin Shearing Buffer and sonicated using Bioruptor Plus (Diagenode) on high power to obtain fragments of 250-300 bp. ChIP reaction mixes containing sheared chromatin, Chromatin Dilution Buffer, anti-CTCF antibody (Millipore, 07-729, lot # JBC1903613 or no-antibody for negative control) and protease inhibitor cocktail were incubated rotating overnight at 4oC. The next day, ZymoMag Protein A beads were added to the mix and incubated for 1 h at 4oC. The complexes were washed with Washing Buffers I, II and III and then the beads were re-suspended in DNA Elution Buffer. Following de-crosslinking with Proteinase K at 65oC, the ChIP DNA was purified using the ZymoSpin IC columns. The samples were stored at -80oC. The concentration of DNA in the ChIP samples was measured using the NanoDrop 3300 fluorospectrophotometer (Thermo Scientific) along with the Quant-iT™ PicoGreen ds DNA assay kit according to the manufacturer’s instructions.
GEO Accession
GSM2731542