GSM9476415: CD4 T cell control donor 1 replicate 2; Homo sapiens; OTHER
Qiagen DNeasy blood and tissue kit 7-10 µg of each 3C library was sonicated to a mean fragment size of 200 bp using a Covaris S220 Focussed Ultrasonicator (1-3 cycles of 60s: duty cycle 10%; intensity 5; cycles per burst 200). The library size was assessed using an Agilent TapeStation (DNA 1000) and an Ampure XP (Beckman Coulter) bead clean-up was performed once the sample had been adequately sonicated. To maximise library complexity three aliquots, each containing 2 µg sonicated 3C library, were processed in parallel for each sample to add sequencing adaptors. NEB Ultra II reagents were used following the standard protocol with the following modifications: first, additional adapter was added into the ligation reactions (7.5 µl rather than 2.5 µl); no size selection was performed (Ampure XP bead clean ups were always performed with a sample:bead ratio of 1:1.8) and the final PCR to add indices was amplified in triplicate from each aliquot using the Herculase II PCR kit (Agilent) to maximise DNA yield. All parallel library preparations and PCR reactions were pooled for each reaction. CHIP libraries were prepared and analyzed as previously reported in Hanssen LLP. et al., 2017.