GSM9415270: ChIP, H2A, SSRP1-FKBP-E14, dTAG-13 3h, clone13; Mus musculus; ChIP-Seq
2.5M cells were fixed with 1% FA for 10min. Fixed cells were lysed with an SDS lysis buffer (1% SDS, 10 mM Tris-HCl pH 8.0, 1 mM EDTA) and via passage through a 27G needle. Samples were sonicated (Covaris ME220) aiming for 200–300 bp fragments. Sonicated chromatin from Drosphila (S2 fixed cells) was spiked in a ratio of 4:1 (mouse:dros) To reduce non-specific binding, samples were pre-cleared with Protein A and G-coupled dynabeads (1:1). Five percent of the sample was separated as an input, and the rest was incubated with the corresponding antibody overnight. Antibody-chromatin complexes were isolated with Protein A- and G-coupled dynabeads, washed with RIPA (3 washes), Tris-EDTA (1 wash) and eluted with SDS lysis buffer. Samples were treated with RNase, Proteinase K and kept at 65C overnight to reverse crosslinks. DNA was then purified with the Monarch PCR & DNA Cleanup Kit (T1030) The protoocol for NEBNext Ultra II library prep kit (E7645) was followed as indicated by the manufacturer.