day 5 of Spinal Motor Neuron in vitro directed differentiation
background
129/Ola
chip antibody
H3K27me3 (Millipore 07-449)
differentiation
ESCs were subjected to Spinal motor Neuron in vitro directed differentiation as previously described (Wichterle and Peljto, 2008). Cells were collected at day 5.
Sequenced DNA Library
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
Chromatin immunoprecipitation (ChIP) was performed based on the protocol as previously described (Lee et al., 2006), with modifications and adaptations. Briefly, a Diagenode bioruptor was used for sonication of formaldehyde-crosslinked cells on high for 30s on/ 30s off for 45 cycles in sonication buffer (20mM Tris-HCl; 150mM NaCl; 2mM EDTA; 0.1mM SDS; 1% Triton X-100; protease inhibitor (Thermo-scientific)). The Diagenode IP-star was also used for automation of the ChIP protocol in all but ST7_Suz12GT_K27, according to the manufacturer’s specifications. After purification of DNA, samples were used for to prepare libraries for Illumina sequencing. Library preparation is performed essentially as previously described (Schmidt et al., 2009); the amplification and size selection steps are reversed in order, and size selection was performed using Agencourt Ampure XP beads. Sequencing was run on an Illumina Hi-Seq (barcoded).