title: GSM3211743: GM04025_CTCF_Input_Rep2; Homo sapiens; ChIP-Seq
library_construction_protocol: 20 million B-lymphocytes were fixed in serum-free RPMI 1640 in 1% formaldehyde for 10 minutes at room temperature. Fixation was terminated by adding glycine to a final concentration of 0.2 M and incubating for 5 minutes at room temperature. Cells were spun down for 5 minutes at 1,350 rpm at 4C, the supernatant removed and washed once with PBS.  Cells were lysed by incubating on ice for 10 minutes in 1 mL Cell lysis buffer (10 mM Tris pH 8.0, 10 mM NaCl, 0.2% NP-40/Igepal, Protease Inhibitor, PMSF). Lysed cells were pelleted for 5 minutes at 300xg at 4C and the resulting nuclei were lysed by re-suspending in 500 mL Nuclear Lysis Buffer (50 mM Tris pH 8.0, 10 mM EDTA, 1% SDS, Protease Inhibitor, PMSF) and incubated on ice for 20 min. Samples were sonicated after adding 300 μl IP Dilution Buffer (20 mM Tris pH 8.0, 2 mM EDTA, 150 mM NaCl, 1% Triton X-100, 0.01% SDS, Protease Inhibitor, PMSF) using a QSonica Q800R2 sonicator for 1 hour set at 100% amplitude, with pulse set to 30 seconds on and 30 seconds off. After sonication, samples were pelleted at 13,000 rpm for 5 min at 4C. The supernatant was transferred to pre-clearing reactions containing 3.7 mL IP Dilution Buffer, 500 μl Nuclear Lysis Buffer, 175 μl of a 1:1 ratio of ProteinA:ProteinG bead slurry and 50 ug of rabbit IgG. Samples were rotated at 4C for 2 hours.  Next, 200 μl was aliquoted from the pre-cleared chromatin as the whole cell extract 'input' control. The remainder of the DNA was added to the pre-bound IP reaction consisting of 1mL cold PBS, 20 μl Protein A, 20 μl Protein G, and 10 μl CTCF antibody. Pre-binding of the IP reaction was performed the previous night by rotation at 4C. Upon addition of DNA, IP reactions were rotated overnight at 4C.   IP reactions were pelleted at 4,000 RPM for 5 minutes at 4C. The supernatant was discarded and the bead pellet was washed once with IP Wash Buffer 1 (20mM Tris pH 8, 2mM EDTA, 50 mM NaCl, 1% Triton X-100, 0.1% SDS), twice with High Salt Buffer (20 mM Tris pH 8, 2mM EDTA, 500 mM NaCl, 1% Triton X-100, 0.01% SDS), once with IP Wash Buffer 2 (10 mM Tris pH 8, 1mM EDTA, 0.25 M LiCl, 1% NP-40/Igepal, 1% sodium deoxycholate) and twice with TE buffer (10mM Tris pH 8, 1mM EDTA pH 8). Washed beads were eluted twice with 100 mL of Elution buffer (100mM NaHCO3, 1%SDS, prepared fresh) by adding 110 μl buffer to each sample, spinning at 7,500 rpm for 2 minutes and removing 100 μl of supernatant as the final eluate. To degrade residual RNA, 2 μl RNAse A and 12 μl 5M NaCl were added to each IP and input sample and incubated at 65C for 1 hour. To reverse crosslinks, 3 μl of proteinase K were added to all samples and incubated overnight at 65C.   To extract DNA, 400 μl of 1X TE and 400 μl phenol:chloroform were added to each sample and vortexed for 60 seconds. Samples were then spun at 14,000 rpm for 10 min at 4C. The top phase was transferred to a new tube and  DNA was precipitated by adding 16 μl 5M NaCl, 6 μl glycogen, 1mL cold 100% ethanol and 40 μl 3M sodium acetate to each sample. Samples were incubated at -80C for 2 hours and then pelleted at 14,000 RPM for 1 hour at 4C. Pellets were resuspended in 1 μl cold 80% ethanol and spun for 30 min at 14,000 RPM at 4C. Supernatent was removed and pellets were dried on 37C heat block for 15 minutes. DNA pellets were dissolved in 20 μl 1X TE at 37C for 15 minutes. Libraries were prepared for sequencing using the NEBNext Ultra II Library Prep Kit for Illumina following the manufacturers protocol. 0.5 ng of DNA was used for starting material, which was quantified using Qubit dsDNA HS Assay Kit. No size selection step was performed after adaptor ligation. Libraries were amplified over 11 PCR cycles using NEBNext Multiplex Oligos for Illumina. High sensitivity electrophoresis was performed on an Agilent 2100 Bioanalyzer to confirm the library size of 250 to 1200 bp. Library concentration was assayed via the KAPA Illumina Library Quantification Kit and diluted to equivalent concentrations. Pooled libraries were sequenced using a 75-cycle paired-end kit on a Illumina NextSeq 500.
attributes.tag: GEO Accession
attributes.value: GSM3211743