title: GSM8865454: Hela cells, Cx46 overexpression, Chromatin input; Homo sapiens; ChIP-Seq
library_name: GSM8865454
library_construction_protocol: Nuclear extracts were made from HeLa cells transfected with pCAG-Cx46, a total of 2e107 cells were washed with PBS and crosslinked with 1% Formaldehyde under gentle agitation at RT after 10 minutes glycine was added at a final concentration of 0.125M and incubated for an additional 10 minutes under agitation. The cells were washed with PBS 3 times and collected with the help of a cell scraper. Then the pellet was resuspended in 5 volumes of lysis buffer (50mM HEPES pH: 7.8, 3mM MgCl2, 20mM KCl, 0.1% NP-40, 2mM phenylmethylsulfonyl fluoride (PMSF), Aprotinin 2 µg/mL, Leupeptin 2 µg/mL and Pepstatin 1 µM) and incubated for 10 minutes on ice. Purified nuclei were obtained with a glass Dounce and verified by Trypan Blue. Nuclei were centrifuged and resuspended in Sonication Buffer (50 mM HEPES pH: 7.8, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% Sodium Deoxycholate, 1% Sodium Dodecyl Sulfate, 2mM PMSF, Aprotinin 2 µg/mL, Leupeptin 2 µg/mL and Pepstatin 1 µM) and transferred to Bioruptor sonication tubes and sonicated for 50 minutes at 30 second intervals on a Bioruptor Pico sonication device. The degree of chromatin fragmentation was verified on a 1% agarose gel by decrosslinking an aliquot in a thermocycler at 95°C for 25 minutes, then 2 µg/mL of RNase A was added and incubated at 37°C for 15 minutes and finally Proteinase K 50 µg/mL was added and incubated for 15 minutes at 58°C. DNA was quantified using the Qubit dsDNA BR Assay Kit on a Qubit Fluorometer following the manufacturer's instructions Libraries were prepared wit MicroPlex Library Preparation Kit v3  (Diagenode) unfer recommended protocol
identifiers.namespace: GEO
identifiers.id: GSM8865454_r1