GSM2367730: Input for ChIP Aly-HA from Aly-HA; kmg KD rep2; Drosophila melanogaster; ChIP-Seq
85 pairs of testes were dissected at a time in PBS. Dissected testes were fixed in 1% formaldehyde in PBS at room temperature for 8 minutes in rotating test tubes. The reaction was stopped by adding 2.5M glycine to the testes in 1% formaldedhyde to a final concentration of 125mM glycine and incubating at 4°C for 5 minutes. Then the fixed testes were washed in PBS three times and snap frozen in liquid nitrogen, and stored in -80°C. Eight such batches (680 pairs altogether) were used per condition. After accumulating enough samples, each batch of 85 pairs of frozen, fixed testes were thawed and homogenized using a plastic pestle, and resuspended in 130uL of sonication buffer (10mM Tris pH 8.0, 1mM EDTA, 0.1% SDS). The testis lysates were transferred to Covaris sonication miniTubes (Covaris, microTUBE AFA Fiber Pre-Slit Snap-Cap 6x16mm), and sonicated for 10 minutes each in the Covaris E220 Focused-ultrasonicator (Treatment Time: 600 sec; Acoustic Duty Factor: 2 %; Peak Incident Power: 105 W; Cycles Per Burst: 200) to a final DNA size distribution maximum between 300-500bp. After sonication, lysates from all 680 pairs of testes for the same genotype were pooled together and centrifuged at 4ºC for 5 minutes to clear debris. About 40 µL (~4%) of supernatant was saved to check fragment size and also to use as an input sample. 1/3 volume of 3X IP buffer was then added to sonicated testes lysates to achieve a final buffer composition of (20mM Tris pH 8.0, 135mM NaCl, 10% glycerol, 1% NonidetP-40, 5mM EDTA, 0.03% SDS, 1mM PMSF, 1x Complete protease inhibitor (Roche, Cat# 11697498001)). Testis lysates in IP buffer were incubated with Protein A magnetic beads conjugated with antibodies against Kmg, HA (for Aly) or dMi-2, prepared as described above for protein immunoprecipitation. After overnight incubation, magenetic beads were washed two times with IP buffer (without SDS), two times with high salt wash buffer (same as the IP buffer but with 270mM NaCl), two times with NaDOC buffer (10mM Tris, pH 8.0, 250mM LiCl, 0.5% Nonidet P-40, 1mM EDTA, 0.5% NaDOC), and finally one time with TE NaCl (10mM Tris pH 8.0, 1mM EDTA, 50mM NaCl), then eluted with 200µl elution buffer (50mM Tris pH 8.0, 10mM EDTA, 1% SDS, 1mM PMSF, 1x Complete protease inhibitor (Roche, Cat# 11697498001)) twice by incubating at 70ºC with occasional mixing every five minutes for 30 minutes. Immunoprecipitated protein-chromatin complexes were treated with RNase A for 30 minutes at 37ºC, then with proteinase K for three hours at 55 ºC, then de-crosslinked by incubating at 65ºC for >12 hours. DNA was extracted from de-crosslinked eluates by phenol:chloroform:isoamyl alcohol (25:24:1) based phase separation followed by ethanol/sodium acetate precipitation. Precipitated DNA was resuspended in 15 µl of 10mM Tris, pH 8.0. The concentration of purified DNA was measured by Qubit (dsDNA HS Assay kit, Thermo Fisher Scientific, Cat#: Q32851). Because the amount of immunoprecipiated DNA was very low (~1ng), sequencing libraries were generated without a purification step until the final PCR amplification by using a kit utilizing stem-loop adapters (Rubicon genomics, ThruPLEX DNA-Seq 12S kit) based on the manufacturer’s instructions. All ChIP-Seq libraries were sequenced by NextSeq High Output mode with 75bp single end, except one replicate of the Kmg ChIP-Seq, which was sequenced with NextSeq Mid Output mode with 75bp paired end. Between 4 to 8 libraries were pooled into one sequencing lane, on average yielding over 40 million reads per sample (17 ~ 81 million). All sequencing was performed by the Stanford Functional Genomics Facility (SFGF).
GEO Accession
GSM2367730