GSM7660960: Cl13Stat5CA_R2; Mus musculus; OTHER
Reaction was stopped by addition of 150microL of 2X stop Buffer (340mM NaCl, 20mM EDTA, 4mM EGTA, 0.02% Digitonin, 50microg/ml RNAseA and 50microg/ml Glycogen) followed by 10min incubation at 37oC to release target chromatin. Samples were then centrifuged 5min 16,000g 4oC and supernatants were transferred to new tubes. Chromatin fragments were incubated 10min at 70oC with 3microL of 10% SDS and 2.5microL of proteinase K (20mg/ml) followed by phenol/chloroform/isoamyl alcohol-based extraction according to original protocol (method B). Upper phase containing DNA was mixed with 1microL of glycogen (20mg/ml) and incubated with 750microL of cold 100% ethanol at -20oC O/N. Samples were centrifuged 30min 16,000g 4oC, rinsed once with 1ml of cold 100% ethanol and centrifuged again for 5min 16,000g 4oC to remove residual ethanol. Samples were air-dried, resuspended in 50microL of molecular grade water and stored at -20oC. DNA libraries were built using the NEBNext Ultra II DNA Library Prep Kit for Illumina (NewEngland Biolabs) with the following modifications.2 NEBNext End Prep step was performed using 25microL of input material for a final volume of 30microL and the following adapted program (30min-20oC, 60min-50oC, Hold at 4oC). Adaptor was diluted at 1:25 and added at 1.5microL for ligation (15min-20oC) followed by addition of 1.5microL of Red USER Enzyme and additional 15min incubation at 37oC. Size selection was performed using 80microL of AMPure XP beads (Beckman Coulter) and purified DNA fragments were amplified for 14 cycles (annealing time changed to 10s). Libraries were cleaned-up with two rounds of size selection with AMPure XP beads (24microl/12microl; Beckman Coulter) eluted in 15microl of molecular grade water, and amplicons quality was assessed on a 2200 TapeStation (Agilent Technologies) Libraries were quantified by qPCR using the NEBNext Library Quant kit for Illumina (NewEngland Biolabs) according to manufacturer's protocol and pooled at equal molarity (1nM).  Denatured Libraries were diluted at 1.8pM, loaded into a NextSeq 500/550 High Output Kit (75 cycles, Illumina) and paired-end sequencing was performed on a NextSeq 550 (Illumina).