GSM8171998: Background_control_Bcells_WT_rep1_72h_post_activation; Mus musculus; OTHER
After 72h h, the culture medium was removed, and cells were washed once with cold PBS and twice with TE buffer (10 mM Tris [pH 8.0] and 1 mM EDTA). Cell lysis was performed directly on the plate by adding 0.5 ml of lysis buffer (TE supplemented with 0.625% SDS and 0.0625 mg/ml 7 Proteinase K), and the suspension was immediately transferred to a fresh 2 ml tube and incubated for 4.5 h at 37°C with gentle mixing every 15 min. genomic DNA was purified by phenol:chloroform:isoamyl alcohol (25:24:1) extraction and ethanol precipitation. Precipitated DNA was washed with 70% ethanol, air-dried, and resuspended in 30 µl of TE buffer. Next, pure gDNA was sheared by sonication using Bioruptor Plus with a water-cooling system (Diagenode) as the following: 3 µg gDNA diluted to 200 µl in TE was placed in 1.5 ml DNA LoBind Tubes (Eppendorf) and subjected to 30 cycles of sonication (30 s ON, 90 s OFF) in “Low” mode. The length of fragments after sonication was evaluated by running DNA samples before and after shearing on a 1.5% agarose gel in 1X TAE (40 mM Tris [pH 8.0], 20 mM acetic acid, and 2 mM EDTA) at 70-80 V. To ensure specificity, nucleic acids are pre-treated with Ribonuclease H (RNase H) (NEB, cat. No. M0297S) to eliminate RNA:DNA hybrids from the mixture. In this process, 4 μg of digested gDNA is incubated with 4 μL of NEB RNase H for 4 hours at 37°C. Following this treatment, proceed with the S9.6 immunoprecipitation step, processing both the control, RNase H-treated samples alongside the experimental samples. Afterward, 4 µg of fragmented gDNA was mixed with 10 µg of S9.6 antibody, diluted to a final volume of 1 ml with binding buffer (20 mM HEPES [pH 7.4], 150 mM NaCl, and 0.05% Triton X-100), and incubated overnight with gentle rotation in a refrigeration cabinet. The next day, input fractions that contained S9.6 were added to 50 µl of M2 anti-FLAG Magnetic Beads (Sigma) or Protein G Dynabeads (Thermo), respectively, and incubated for 2 h. Additionally, a second set of negative controls were prepared by incubating 4 µg of sonicated gDNA with beads without the bait. Following incubation, the unbound fraction was removed, and beads were washed three times with 500 µl of binding buffer for 5 min with gentle rotation in a refrigeration cabinet. Elution was performed by adding 125 µl of elution buffer (50 mM Tris [pH 8], 1 mM EDTA, 0.5% SDS, and 0.64 mg/ml Proteinase K) for 45 min at 55°C. Two replicates of each sample were pooled and purified using a DNA clean and concentrator kit (Zymo Research) according to the manufacturer's protocol. After elution with 30 µl of elution buffer (Zymo Research), half of the eluate was treated with 2 µl of DNAse I (1 U/µl) in 1X DNase I digestion buffer (Thermo Fisher Scientific) for 1 h at 37°C. For DNase I inactivation, 1 µl of 0.5 M EDTA was added for 10 min at 65°C. RNA was purified using the RNA clean and concentrator kit (Zymo Research). The purified RNA underwent NGS library preparation and subsequent sequencing, which was conducted commercially by Azenta Life Sciences using their strand-specific protoco