title: GSM4306339: pol2_PTPBP1; Homo sapiens; ChIP-Seq
library_construction_protocol: Approximately 10×106 cells per sample were trypsinized, washed with PBS, and crosslinked with 1% FA at 37 °C. After incubation for 10 min, the reaction was stopped by addition of glycine (0.125 M) and incubated at 37 °C for 5 min. Samples were centrifuged at 2300 x g, washed with cold PBS, and centrifuged again. Nuclei isolation, MNase (Worthington) digestion, and IP were done as described (Kfir et al. 2015) with the following minor adaptations: After suspension in IP buffer, the samples were sonicated using a Bioruptor (Diagenode) at 40% amplitude in intervals of 1.5-sec pulses with 9.9-sec pauses for 10 min. For the double ChIP-seq, ChIP was performed as described previously (Yearim et al. 2015) with the following adaptations: Approximately 7×106 HEK293 cells were used per sample. After nuclei purification and MNase digestion, samples were sonicated using a Bioruptor at 40% amplitude with intervals of 2.2-sec pulses with 9.9-sec pauses for 12 min. For anti-pol II p-Ser2 IP, 80 µl of a mixture of protein A and G Dynabeads (Invitrogen) were used with 18 µg anti-pol II p-Ser2 antibody (Abcam; ab5095). After IP, 1 µl of 10 mg/ml RNase A (Sigma) was added, and samples were incubated for 30 min at 37 °C. Following washes, the samples were eluted with 50 μl fresh 0.1 M DTT. As previously described (Sadeh et al. 2016), 50 µl of freshly prepared 2X Chromatin Release Buffer (500 mM NaCl, 2% deoxycholate, 2% SDS, 2 mM EDTA) with fresh EDTA­free protease inhibitor cocktail and PMSF (from the RNA ChIP-IT kit) were added, and samples were incubated at 37 °C for 55 min. The elution step was repeated and samples were incubated for 30 min at a temperature higher than 15 °C to prevent SDS precipitation. This step releases bound chromatin and inactivates the antibodies used in the first ChIP. To the eluted samples 1.5 µl Proteinase K (NEB) was added, and samples were incubated for 16 h at 65 ºC. DNA was purified using phenol:chloroform:isoamyl alcohol (Sigma) extraction. For the second ChIP, 50 µl of a mixture of protein A and G Dynabeads (Invitrogen) were used with 10 µg anti-U1C antibody (Abcam; ab157116). After incubation for 16 h at 4 ºC, the samples were washed and eluted as described previously (Yearim et al. 2015). Deep sequencing libraries were prepared using Illumina TruSeq library preparation kits as per the manufacturer's instructions. Sequencing of 50-bp single-end reads was performed using an Illumina HiSeq 2000.
attributes.tag: GEO Accession
attributes.value: GSM4306339