GSM3576645
GSM3576645: Wt_5K_H3K4Me3_R1; Drosophila melanogaster; OTHER
Conventional libraries: After precipitating and pelleting, DNA was dissolved in 30 μl of TE buffer pH 8.0. The recovered DNA was converted into libraries using NebNext Ultra II DNA library preparation kit, following manufacturer's protocol. TAF-ChIP: Fixed cells were directly sorted in 240 μl of 140mM RIPA (10mM Tris-Cl pH 8.0, 140mM NaCl, 0.1mM EDTA pH8.0, 1% Triton X-100, and 0.1% SDS), and sonicated with 3 cycles at low power settings for breaking the nuclei. 15 μl of Protein A and G Dynabeads were coupled to 1 μg of specific antibody in the blocking buffer (RIPA 140mM supplemented with 0.2 mg/ml BSA, 0.05 mg/ml of glycogen and 0.2 mg/ml of yeast t-RNA) for 3 hrs at 4°C. The unfragmented chromatin were centrifuged at 14000 rpm for 10 minutes at 4°C, and the supernatant was transferred to the tube with blocked and antibody coupled beads. The samples were incubated at 4°C overnight with head over tail rotations. The samples were then washed twice briefly with 300 μl of home made tagmentation buffer (20 mM Tris(hydroxymethyl)aminomethane pH 7.6; 10 mM MgCl2; 20% (vol/vol) dimethylformamide) using magnetic rack for beads separation. The washed beads were resupended in 20 μl of 1X tagmentation DNA buffer (Nextera XT Kit) containing 1 μl of Nextera DNA tagmentation enzyme and incubated at 37 °C for 40 minutes with constant shaking in a thermoblock at 500 rpm. Following the tagmentation, the beads were washed as following; once with 140mM RIPA (10mM Tris-Cl pH 8.0, 140mM NaCl, 0.1mM EDTA pH8.0, 1% Triton X-100, and 0.1% SDS), four times with 250mM RIPA (10mM Tris-Cl pH 8.0, 250mM NaCl, 0.1mM EDTA pH8.0, 1% Triton X-100, and 0.1% SDS) and twice with TE buffer pH8.0 (10mM Tris-Cl pH 8.0 and 0.1mM EDTA pH8.0). The samples after proteinase K treatment for reverse-crosslinking were subjected to phenol chloroform extraction. After precipitating and pelleting, DNA was dissolved in 30 μl of TE buffer pH 8.0. The DNA was amplified in a 100 μl reaction with 1X NEBNext High-Fidelity PCR Mix with primers containing molecular indices (listed in Table 1) with following program; 72 °C for 3min, {98 °C for 10 seconds, 63 °C for 30 seconds, 72 °C for 30 seconds} for 12 cycles, 72 °C for 5 minutes, and hold at 4 °C. The PCR reaction was purified with beads based size selection to remove any fragment that might be of larger than 1000 bp size. Ampure Xp beads were added to the PCR reaction in a ratio of 0.2X ratio to bind larger fragments. The magnetic beads were separated with the help of magnetic rack and supernatant was transferred to a fresh tube. Ampure Xp beads were added to the PCR reaction in a ration of 0.8X to bind the target library. After PCR purification, libraries were analyzed on Agilent Bioanalyzer for size distribution and the concentration was measured using Qubit fluorometer. The finished libraries were pooled in equimolar amounts and sequenced on illumina NextSeq 500.
GEO
GSM3576645
GEO Accession
GSM3576645