GSM7835834: D17 cells, ChIP-seq, Ph, replicate 1; Drosophila melanogaster; ChIP-Seq
For each cell type started with 30 mL of cells at 6106 cells/ml. Added 1.87 mL of 16% formaldehyde and incubated at RT for 10 min with gentle rocking. The formaldehyde was quenched by adding glycine to a final concentration of 0.125 M followed by a 5 min incubation at RT with gentle rocking. The cells were spun down at 2000 g, 3 min, and washed twice with ice cold PBS. The cells were resuspended in 600 µL PBS, divided into 100 µL aliquots, spun down and the pellets were flash frozen and stored at -80 ℃. Each aliquot of cells can be used for up to 5 ChIP reactions. Cells were fixed and frozen from different flasks to generate independent biological replicates. The frozen formaldehyde fixed cell pellets were resuspended in 0.8 ml ice-cold cell lysis buffer (5 mM PIPES pH 8, 85 mM KCl, 0.5% NP40, supplemented with protease inhibitors (Roche Complete EDTA-free protease inhibitor), incubated on ice for 10 min, then pelleted by centrifugation at 2000 g for 5 min at 4 ℃. The supernatant was removed and the pellet resuspended in 1 mL nuclear lysis buffer (50 mM Tris-HCl pH 8, 10 mM EDTA, 0.2% SDS, supplemented protease inhibitors) and incubated for 10 min at 4 ℃ on a rocking platform. 0.5 ml of 400 mM NaCl IP dilution buffer (16.7 mM Tris-HCl pH 8, 1.2 mM EDTA, 400 mM NaCl, 1.1% Triton X100, 0.01% SDS, supplemented with protease inhibitors) – to a final salt concentration of 125 mM. The lysate was sonicated in 300 µL aliquots using a Q Sonica (Model Q800R3), at 40% amplitude for 30 sec OFF and 30 sec ON for a total of 5 min ON time. The sonicated samples were spun for 10 min at full speed in an Eppendorf centrifuge at 4 ℃. The 300 µL aliquots of each cell type sample were pooled again. 4 µL was removed as input (2% of a single ChIP reaction). To the remainder 100 uL of TE washed Protein A SepharoseTM Fast Flow (Cytiva) was added, and the samples were incubated at 4 ℃ for 1 hour with gentle rocking. The samples were spun at full speed in an Eppendorf centrifuge for 1 min, and the supernatant transferred to a fresh tube. For each IP, 200 uL of sonicated the sample was used + 800 uL 67 mM NaCl ChIP dilution buffer (16.7 mM Tris-HCl pH 8, 1.2 mM EDTA, 67 mM NaCl, 1.1% Triton X100, 0.01% SDS, supplemented with protease inhibitors. The appropriate amount of antibody (Supplemental Table 1), was added and incubated rocking overnight at 4 ℃. 60 µL protein A/agarose bead slurry (prewashed with TE buffer prior to use) for 1 hour at 4 ℃ with rotation. The agarose was pelleted by centrifugation at 800 rpm at 4 ℃ for 1 min, the supernatant was carefully removed and the agarose was washed 5 min on a rotating platform at 4 ℃ sequentially with 1ml of the following buffers: Low salt immune complex buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8.0, 150 mM NaCl), High salt immune complex wash (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.0, 200 mM NaCl), LiCl immune complex wash (0.25 M LiCl, 1% NP40, 1% deoxycholic acid (sodium salt), 1 mM EDTA, 10 mM Tris pH 8.0) followed by two 5 min room temperature washes with TE buffer. The agarose was pelleted again at 800 rpm for 1 min and the DNA was eluted with 500 µL freshly made elution buffer (1% SDS, 0.1 M NaHCO3) for 30 min with gentle rocking. The agarose was pelleted and the supernatant transferred to a fresh tube. The crosslinks were reversed by adding 20 µL of 5 M NaCl and incubating at 65 ℃ for 4 hours. The cross links were also reversed in the input sample. 10 uL of 0.5 M ETDA, 20 uL of 1 M Tris-HCl pH 6.5 and 2 uL of 10 mg/mL proteinase K were added and the samples incubated at 45 ℃ for 1 hour. The DNA was recovered by phenol/chloroform extraction followed by ethanol precipitation with 2 µL of pellet paint, 50 µL 5 M NaOAc, 1 mL Ethanol. Washed and dried pellets were resuspended in PCR grade water. 1.5 ng of ChIP sample DNA (as measured by a Qubit 3.0 fluorimeter) was used to prepare each library. Libraries were made using either the Thruplex DNA-seq and dual index kits (Takara), or the NEBNext UltraTM II DNA library preparation kit and dual index kits (New England Biolabs). Libraries were made according to the manufacturer's directions. Samples were sequenced by 50 bp pair-end sequencing with a NovaSeq 6000 with an SP100 kit.by the NICHD Molecular Genomics Core.