GSM5456904: SUDLH5_EV_RAD21_ChipSeq; Homo sapiens; ChIP-Seq
Cells were crosslinked with 1% formaldehyde for 5 min. 6% murine T-lymphoma cells were added during cell lysis as exogenous spkie-in for normalization. After cell lysis, nuclei were re-suspended in RIPA buffer (10 mM Tris/HCl pH 7.5, 150 mM NaCl, 1% NP40, 1% deoxycholic acid (DOC), 0.1% SDS, 1 mM EDTA) and DNA was fragmented to a size <300 bp using a Branson sonifier or using the Covaris Focused Ultrasonicator M220 for 100 min per ml lysate. Chromatin was immunoprecipitated with 15 µg of corresponding antibody for 6 to 8 hours. Chromatin was eluted with 1% SDS and crosslinking was reverted overnight. For purification, chloroform/phenol extraction was used. NEBNext® ChIP-Seq Library Prep Master Mix Set for Illumina® or NEBNext® Ultra II DNA Library Prep with Sample Purification Beads (New England Biolabs). Purified DNA was end-repaired, A-tailed, ligated to Illumina adaptors, size-selected (200 bp) and purified with Qiagen gel extraction kit or Sample Purification Beads. DNA fragments were amplified by 17 cycles of PCR and library size was tested with the Fragment Analyzer (Advanced Analytical). The amount of library DNA was quantified using a picogreen assay.
GEO
GSM5456904