title: GSM4183167: SA1-AID no aux SMC3 ChIP; Homo sapiens; ChIP-Seq
library_construction_protocol: ChIP-Seq for SMC3 was performed as described (Cabianca, Casa et al. 2012) with some adaptations. Approximately 5*107-1*108 cells were crosslinked by replacing the medium with the same volume 1% formaldehyde in PBS  for 10' at RT. After quenching with 0.125M Glycine (final concentration), cells were washed three times with PBS, harvested using a silicon scraper. Lysis of the cells was carried out by incubating the cells in LB1 buffer (50 mM Hepes-KOH, pH 7.5, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0,5% NP-40, 0,25% Triton X-100) for 5', followed by centrifugation at 1,350g for 5 min and incubation with LB2 buffer (10 mM Tris-HCl, pH= 8.0, 200 mM NaCl, 1 mM EDTA, 0,5 mM EGTA) for 10'. Nuclei were pelleted down by centrifugation of the cells at 1,350g for 5 min and were resuspended in LB3 buffer (10 mM Tris-HCl, ph=8,0, 100 mM NaCl, 1 mM EDTA, 0,5 mM EGTA, 0,1% Na-Deoxycholate, 0,5% N-lauroylsarcosine). All lysis buffer contained 1x protease inhibitors. This protocol ensures the removal of cytoplasmic proteins from the lysates. Sonication was performed using a Diagenode bioruptor (15 min, 5-7 cycles, 30” each). Sonicated lysates were cleared by the addition of Triton X-100 to a final concentration of 1% and by centrifugation at maximum speed for 10 min at 4°C. 50 µl of the sonicated lysates were reserved for input and the remaining material stored at – 80°C until use. To prepare the antibody-loaded beads, 100 µl of Dynabeads were washed three times with blocking solution (1x PBS, 0,5% BSA) and incubated overnight with 10 µg of the required antibodies. For each ChIP 100 µg  chromatin was incubated overnight at 4°C with the antibody-bound beads. Beads were washed 6X with RIPA buffer (50 mM Hepes-KOH, pH= 7,6, 500 mM LiCl, 1 mM EDTA, 1% NP-40, 0,7% Na-Deoxycholate), 1X with TE/50mM NaCl. The DNA was eluted from the beads by incubation with 250 µl of elution buffer (1x TE , 2% SDS) for 15 min at 65°C. Crosslinking of the ChIP and input samples was reversed by incubation at 65°C overnight, proteins were digested  by incubation with 8 µl of 10 mg/ml of Proteinase K at 55°C for 1 hour and RNA degraded by incubation with 8µl of 10 mg/ml  at 37°C for 30 min. DNA was purified then purified using phenol-chloroform extraction followed by ethanol precipitation. The DNA was then resuspended in 25 µL TE and used sequencing. The samples were prepared as Chip Seq Multiplex samples using the NEXTFlex ChIP-Seq kit from BioO Scientific and sequenced them according to the Illumina TruSeq v3 protocol on the HiSeq2000 for a single read 50bp in length and a 7bp index read. The data were de-multiplexed and mapped against GRCh38/hg19 as indicated below. NGS libraries have been prepped from the ChIP DNA using the NEXTFlex ChIP-seq kit from BioO Scientific and sequenced according to the Illumina TruSeq Rapid v2 protocol on the HiSeq2500 for a single read 50bp in length and a 8bp dual index read.
attributes.tag: GEO Accession
attributes.value: GSM4183167