title: GSM6940629: RTK04 - Targeting biol rep 1 ChIP; Homo sapiens; ChIP-Seq
library_name: GSM6940629
library_construction_protocol: 72 hours after transfection, cells were washed with DPBS with no calcium or magnesium (Fisher Scientific), harvested using TrypLE (Fisher Scientific), and neutralized with culture media. The resuspended cells were pelleted by centrifugation at 300 x g for 5 minutes, and the supernatant was aspirated. Pellets were resuspended in 1% freshly made formaldehyde (Thermo Fisher Scientific) in DPBS and shaken gently for 10 minutes. Fixation was quenched by adding 2.5 M glycine, for a final concentration of 125 mM glycine, and shaking cells gently for 5 minutes. Cells were pelleted as described above, washed with cold DPBS, pelleted, resuspended in DPBS and 1x cOmplete EDTA-free protease inhibitors (Millipore Sigma), pelleted, flash frozen in liquid nitrogen, and stored at -80 °C. On the day of sonication, the cross-linked pellets were resuspended in 1 mL of Lysis Buffer 1 (50 mM HEPES-KOH, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% Triton X-100) and 1X protease inhibitors and rotated for 10 minutes. Cells were pelleted at 1350 g for 5 minutes. Pellets were resuspended in 1 mL of Lysis Buffer 2 (10 mM Tris-HCl, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA) and 1X protease inhibitors and rotated for 10 minutes before being pelleted at 1350 g for 5 minutes. Pellets were resuspended in 900 uL of Lysis Buffer 3 (10 mM Tris-HCl, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% Na-Deoxycholate, 0.5% N-lauroylsarcosine), 100 uL of 10% Triton X-100, and 1X protease inhibitors. All steps took place at 4 °C. The resuspended cells were transferred to 1 ml milliTUBE AFA Fiber (Covaris) and sonicated on M220 Focused-ultrasonicator (Covaris) under the following SonoLab 7.2 settings:  minimum temperature 4°C, set point 6 °C, maximum temperature 7 °C, Peak Power 75.0, Duty Factor 10.0, Cycles/Burst 200, sonication time 490 seconds. Sonicated cell lysate was centrifuged at 20,000 g for 10 minutes at 4 °C. The supernatant was transferred to a new tube, and 5% was saved as the input sample. The remaining supernatant was incubated with Dynabeads Protein G (Thermo Fisher Scientific) that were bound to the monoclonal anti-Flag M2 antibody (Sigma-Aldrich) the day before sonication by overnight rotating at 4 °C, and the lysate-Dynabead mixture was rotated overnight at 4 °C. The samples were washed three times each with low salt buffer (150 mM NaCl, 0.1% SDS, 1% Triton X-100, 1 mM EDTA, 50 mM Tris HCl), high salt buffer (550 mM NaCl, 0.1% SDS, 1% Triton X-100, 1 mM EDTA, 50 mM Tris HCl), and LiCl buffer (150 mM LiCl, 0.5% Na-deoxycholate, 0.1% SDS, 1% Nonidet P-40, 1 mM EDTA, 50 mM Tris HCl) on a magnetic stand at 4 °C. The samples were washed with 1 mL of TE buffer (1 mM EDTA, 10 mM Tris HCl) with 50 mM NaCl and centrifuged at 960 g for 3 minutes at 4 °C. The supernatant was carefully aspirated with a pipette. 210 uL of elution buffer (1% SDS, 50 mM Tris HCl, 10 mM EDTA, 200 mM NaCl) was added to samples and incubated for 30 minutes at 65 °C. Samples were centrifuged for 1 minute at 16,000 g at room temperature, and 200 uL of supernatant was incubated overnight at 65 °C. The input sample was diluted in 150 uL of elution buffer and also incubated overnight at 65 °C. 0.5 uL of 10 mg/mL RNase was added, and samples were incubated for 1 hour at 37 °C. 2 uL of 20 mg/mL Proteinase K were added, and samples were incubated for 1 hour at 55 °C. The DNA was recovered by the QiaQUICK PCR Purification Kit (Qiagen) and DNA was eluted in 50 uL of water for downstream analysis. Sample DNA concentration was determined by the DeNovix dsDNA High Sensitivity Kit. Illumina libraries were generated using the NEBNext Ultra II Dna Library Prep Kit for Illumina (NEB). Sample concentrations were normalized such that 12 ng of DNA in each condition was used for library preparation. The concentration of DNA was determined for pooling using the DeNovix dsDNA High Sensitivity Kit. Illumina libraries were sequenced in paired-end mode on the Illumina NextSeq platforms with automated demultiplexing and adaptor trimming. For each ChIP-seq sample, 75-bp paired end reads were obtained and between 9.5 and 18.9 million uniquely mapped fragments were analyzed.