GSM8635811: Vehicle Treatment Replicate 2; Mus musculus; ChIP-Seq
The samples were used in immunoprecipitation reactions with antibodies against PARP7 (in-house N-term antibody) or rabbit IgG (as a control) with incubation overnight at 4 °C with gently mixing. The next day, Protein A Dynabeads were washed 3 times, they were then added to each IP sample and were left to rotate at 4°C for 3 hours. The samples were then washed 2 times  with each of the following buffers (1) RIPA 0 Buffer (10 mM Tris-HCl pH 7.4, 1 mM EDTA, 0.1% SDS, 1% Triton X-100, 0.1% sodium deoxycholate, and 1x complete protease inhibitor cocktail), (2) RIPA 0.3 Buffer (10 mM Tris-HCl pH 7.4, 1 mM EDTA, 0.3 M NaCl, 0.1% SDS, 1% Triton X-100, 0.1% sodium deoxycholate, and 1x complete protease inhibitor cocktail), (3) LiCl Wash Buffer (10 mM Tris-HCl pH 7.9, 1 mM EDTA, 250 mM LiCl, 0.5% NP-40, 0.5% sodium deoxycholate, and 1x complete protease inhibitor cocktail), and (4) 1x Tris-EDTA (TE). The immunoprecipitated genomic DNA was eluted in SDS Elution Buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCL pH 7.9) and was decrosslinked at 65°C overnight. Supernatant was then collected and was digested with proteinase K and RNase A to remove protein and RNA, respectively. ChIP DNA was then recovered using Qiagen PCR Purification Kit (Cat. No. 28104)and was eluted in DEPC- treated, nuclease-free water. ChIP-seq libraries were generated from three biological replicates for each condition. A total of 10 ng for PARP7 ChIPed DNA, or equivalent amounts input DNA, were used to generate libraries for sequencing. ChIP-seq libraries were generated using NEBNext Ultra II DNA Library Prep Kit (New England Biolabs #E7645) and sequenced on an Illumina NextSeq2000 system by paired end 50bp using NEXTseq 2000 P3-100cycle sequencing kit (20040559). n= 3 biological replicates. Publicly available data from GSE27826 was used for some analysis{Siersbaek, 2011 #131}.