GSM4633479: U266_1_Input; Homo sapiens; ChIP-Seq
OCI-Ly3 and OCI-Ly10 were grown in IMDM with Glutamax™ (Life Technologies™), 10% HIFBS, 50µM betamercaptoethanol. Each batch of chromatin from each cell line were harvested from 2*10^7 cells. Batches were pooled together and distributed in aliquots. Individual CHIP were obtained from 4*10^6 cells.  H929 and U266 were grown in RPMI 1640, 10% HIFBS, 1% L-Glutamine. Each batch of chromatin were harvested from 2*10^7 cells. Batches were pooled together and distributed in aliquots. Individual CHIP were obtained from 4*10^6 cells. Peripheral blood was obtained from healthy donors after informed consent. Mononuclear cells were isolated by Lymphoprep (Axis Shield) density gradient centrifugation. Total B-cells were isolated by negative selection with the Memory B-cell Isolation Kit (Miltenyi Biotec). Memory-enriched B cells fractions were isolated by negative selection following incubation of total, negatively selected B cell fractions with CD23 Biotin and anti-Biotin Microbeads (Miltenyi Biotec) Cells were grown in 24 well plates in Iscove Modified Dulbecco Medium (IMDM) supplemented with Glutamax and 10% heat-inactivated fetal bovine serum (HIFBS, Invitrogen). This was supplementetd from Day 3 onwards with Hybridomax hybridoma growth supplement (11 µl/ml), Lipid Mixture 1, chemically defined and MEM Amino Acids solution (both at 1x). B cells were cultured at 2.5 X 10^5/ml with IL-2 (20 U/ml), IL-21 (50 ng/ml), F(ab')2 goat anti-human IgM & IgG (10 µg/ml) on γ-irradiated CD40L expressing L cells (6.25 X 10^4/well). At day-3 cells were detached from the CD40L L-cell layer and reseeded at 1 X 10^5/ml in media supplemented with IL-2 (20 U/ml) and IL-21 (50 ng/ml). For the G9Ai (small molecule inhibitor UNC0638) experiments, ABCs were treated at day-3 with UNC0638 inhibitor (2 µM). Cells were sampled at the indicated time points without further addition of inhibitor ChIP-seq libraries were prepared using the MicroPlex Library Preparation™ kit (Diagenode) for ChIP, size selected using AMPure XP beads (Beckman Coulter) and run on an Illumina Hiseq 2500