Illumina HiSeq 2500 paired end sequencing; Evaluation of Tn5 protein fusions suitable for GET-seq
Evaluation of Tn5 protein fusions suitable for GET-seq
Cells were kept in culture according to standard tissue culture procedure. Cells were detached from plates by trypsin treatment, harvested and counted by Trypan Blue exclusion. Chromatin was collected following an already published study (doi:10.1172/JCI81040). Briefly, after addition of Complete, EDTA-free Protease Inhibitor Cocktail Tablets (Roche) and the phosphatase inhibitors sodium orthovanadate and NaF, lysates were sonicated with Bioruptor (Diagenode) (300 μL aliquots of lysates were sonicated for 10 min - medium intensity 30 sec on 30 sec off). In order to get 300-500 bp DNA fragments. 10-20 µg of ready-to-use chromatin were used for each ChIP. For each ChIP, 70 µl of Dynabeads protein G slurry (Invitrogen) were washed and incubated with 5 µg of antibody. Samples were purified with QIAquick PCR purification kit (Qiagen), following manufacturer's recommendations.  Reactions were purified using QIAquick Gel Extraction Kit (Qiagen) and eluted in 15 μL of EB buffer. 5 μL of this reaction was used as input for indexing PCR using 2X Kapa HiFi polymerase (Roche) and Nextera indexing primers (N7XX and S5XX). Libraries clean up was performed following standard procedure.