Cultured in RPMI1640 + UltraGlutamine +10percent FBS, 20mM HEPES, 50ug/ml Gentamicin and 50uM beta-ME.
antibody
NA
Sequenced DNA Library
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
For RNA-seq: Total RNA was isolated by use of RNAeasy Micro Kit (Qiagen, Hilden, Germany) according to manufacturer's recommendations. For ChIP-seq: Cells for transcription factor ChIP were fixed at room temperature with DSG for 30 min followed by 1percent formaldehyde/PBS for 10 minutes. Reaction was then quenched by adding glycine to 0.125 M. Cells were washed pelleted and snap-frozen and stored at -80°C until ready for ChIP or used immediately for ChIP. Nuclei for ChIP were isolated by 10 min incubation in Nuclei Isolation buffer (50 mM Tris-pH 8.0, 60 mM KCl, 0.5percent NP40) + protease inhibitor cocktail (PIC) (Roche) on ice. Pelleted nuclei were dissolved in Lysis buffer (0.5percent SDS, 10 mM EDTA, 0.5 mM EGTA, 50 mM Tris-HCl (pH 8))+ PIC and sonicated on a Bioruptor (Diagenode) max power for 30s followed by 30 s rest. Sonication was followed by pelleting of debris and the supernatant was transferred to new tube and chromatin was diluted 5X in Dilution Buffer (1percent Triton, 2mM EDTA, 150 mM NaCl, 20 mM Tris-HCl (pH 8) + PIC). Anti-bodies were hybridized to ProteinA or G Dynabeads® (LifeTechnologies) and added to the diluted chromatin. ChIP was performed over night at 4°C. and subsequently washed (1 time with 500 μl Low Salt Immune Complex Wash Buffer, 1 time with 200 μl High Salt Immune Complex Wash Buffer, 1 time with 200 μl LiCl Immune Complex Wash Buffer, 2 times with 200 μl TE buffer) and eluted for 4 h at 65°C ( 20 mM Tris-HCl, pH 7.5, 5 mM EDTA 50 mM NaCl, 1percent SDS, 100 μg RNase A and 50 μg proteinase K) treated and finally cleaned up using Zymo ChIP DNA Clean & Concentrator before ChIP-seq libary preparation. RNA-seq: Libraries were constructed using NuGEN's Ovation Ultralow Library systems (NuGEN Technologies, San Calros, CA) and were subsequently subjected to 76 cycles of NextSeq500 sequencing (Illumina, San Diego,CA). ChIP-Seq: Libraries were constructed using Illumina Truseq Nano DNA library preparation kit or by addition of Bioo-scientiffic Nextflex barcodes following standard procedures. Libraries were run on the Illumina NexSeq500.