GSM4411943: CTRL1_D6_T2C; Homo sapiens; OTHER
2.5x10e6 cells were used for each time point; the cells were cross-linked using 1% formaldehyde at Room Temperature (RT, 24°C) for 10 min and quenched with 0.125 M Glycine. Subsequently, cells were lysed using cold lysis buffer containing 10 mM Tris-HCl pH 8.0, 10 mM NaCl, 0.5% NP-40 and Complete Protease Inhibitors (Roche). Chromatin was digested with the ApoI restriction enzyme (New Englad Biolabs) (400 U/sample) overnight in a thermomixer (VWR) at 37°C at 900 rpm. The digested products were purified via Phenol:Chloroform. The diluted DNA fragments were ligated with T4 DNA Ligase High Concentration (100 U/sample; ThermoFisher Scientific) overnight at 16°C, and then 30 µl of 10 mg Proteinase K/ml (300 µg) were added and incubated at 65°C for 4 h, followed by 30 µl of 10 mg RNAse A/ml (300 µg) for 1h at 37°C, before proceeding to further purification with Phenol:Chloroform. In total, 6 µg of the resulting chromatin were then linearized using the frequent 4bp-cutting enzyme DpnII (New England Biolabs) (1U/µg of DNA) overnight at 37°C, whilst shaking in a thermomixer at 400 rpm. The day after, the material was precipitated by sodium acetate/ethanol before proceeding to T2C library preparation. T2C Library was prepared using 250 ng of linearized chromatin. The samples were re-buffered to 10 mM Tris-HCl, pH8 by a standard AMPure XP (Agencourt) bead clean-up procedure. The chromatin was sheared to 300-400-bp fragments by a S220 Covaris (Covaris Inc.). Concentration was determined by Quant-it high sensitivity (ThermoFisher Scientific). For each sample 100 ng of sheared chromatin was end-repaired and A-tailed using the Kapa hyper prep kit (Roche) according to the manufacturer's instructions. SeqCap library adaptors were ligated followed by AMPure bead clean up. The pre-capture library was amplified by PCR using KAPA HiFi hotstart readymix for 9 cycles. The amplified pre-capture library was purified by bead clean up and quantified by Bioanalyzer DNA1000 assay (Agilent) according to the manufacturer's instructions. A ZEB2 hg19 based design was ordered at NimbleGen (Roche) with baits located between 143270465 and 150642631 of chromosome 2. A hybridization mixture per sample was prepared with 2 µg pre-capture library, 1 mM HE-index-oligo, 1 mM HE universal oligo, COT Human DNA, AMPure XP reagent and added to 4.5 µl of pre-ordered baits and subsequently hybridized for 16 h at 47°C. Post hybridization the samples were washed according to the instructions in the Nimblegen SeqcapEZ Hypercap workflow (Roche), the  chromatin captured using capture beads. The captured library was amplified by PCR using Kapa HiFi mix and purified by AMPure XP beads. The captured library was quantified by Nanodrop spectrophotometer and the quality was assessed using a Bioanalyzer DNA1000 assay. Finally, the captured T2C libraries were denatured and sequenced on an Illumina HiSeq2500 sequencer as described for RNA-seq but with a custom recipe of 6 dark cycles, followed by paired end 101 sequencing with single index using the rapid v2 chemistry according to manufacturer's instructions (Illumina).
GEO Accession
GSM4411943