title: GSM8504676: antiH3-negative-replicate2-ID_S5; Rattus norvegicus; ChIP-Seq
library_construction_protocol: Rodent tissue samples were diced with a scalpel, washed twice with ice-cold PBS, crosslinked in 1% formaldehyde (15 min in PBS) with frequent vortexing, and quenched with the kit provided formaldehyde quenching solution (Abcam, ab223876; 5 min in PBS) before 2x PBS washes. Crosslinked HeLa cells or rodent tissue was resuspended in 500 µL of Extraction and Lysis Buffer supplemented with protease inhibitors, then treated with 10 passes of both the loose and tight Dounce homogenizer settings, before being incubated for 30 min at RT with 1000 rpm shaking. Chromatin pellets were spun down (10,000 rpm, 15 min) and washed once with milliQ water. Chromatin was then sheared by sonication with a Bioruptor Pico (30s on/30s off, 30 min for tissue), insoluble material was spun down (10,000 g, 10 min) and soluble sheared chromatin was stored at -80 °C. Immunoprecipitation (IP) Buffer (from Chromatin Extraction Kit, ab223876) was added to the sonicated chromatin (200 µL of sonicated chromatin was added to 2 mL of 1x IP Buffer) followed by 2 µL of A:G Dynabeads (pre-rinsed at least three times with IP Buffer). The mixture was incubated at room temperature for 30 min with mixing at 900 rpm before the supernatant was removed after centrifugation (1 min, 1000 g). To reduce background signal, further addition and incubation with A:G Dynabeads (2 µL) was performed. The mixture was centrifuged and the supernatant collected. Aliquots of the supernatant were taken (2 x 50 µL) for inputs. To 1 mL of cleared chromatin, α-HA (132 ng) or α-H3 (248 ng) antibody was added, and the solution was incubated overnight at 4 °C. A:G Dynabeads (10 µL) were then added and mixed for 5 h at 900 rpm. The supernatant was removed on a magnetic stand. The beads were then resuspended and washed twice with RIPA (radioimmunoprecipitation assay) wash buffer (50 mM HEPES, pH 7.6, 500 mM LiCl, 1 mM EDTA, 1% NP-40, 0.7% Na-Deoxycholate) followed by TE (Tris-EDTA) buffer containing 50 mM NaCl (1 mL). The bead solutions were centrifuged (3 min, 1000 g) to remove the TE buffer. The chromatin IP was eluted from the beads with 210 µL of elution buffer (50 mM Tris-HCl, pH 8.0, 10 mM EDTA, 1% SDS; made fresh) and incubation at 65 °C for 30 min at 900 rpm. The samples were centrifuged (1 min, 16000 g) at room temperature and <200 µL of the desired supernatant was collected. The chromatin supernatant was de-crosslinked overnight at 65 °C. Input samples were diluted with elution buffer (1:4) and similarly, incubated at 65 °C overnight. To the decrosslinked chromatin samples, RNase (1 µL) was added and the mixture was incubated for 1 h at 37 °C (500 rpm). This was then followed by addition of Proteinase K and incubation for 1 h at 45 °C (500 rpm). The DNA was recovered by Qiagen PCR purification kit using standard protocol. Qiagen spin columns were used to bind to the DNA. The DNA was then eluted with 50 – 200 µL of H2O. The DNA was prepared for sequencing using the NEBNext® Ultra™ II DNA Library Prep Kit for Illumina® (New England Biolabs, E7645) following the manufacturer's instructions. Briefly, ChIP DNA (~10 ng in 50 µL TE buffer; concentration quantified using a High-sensitivity d1000 TapeStation (Agilent)) were end-repaired, A-tailed and ligated using reagents provided in the kit. Cleanup of the ligation reaction was performed without size selection. The ligated DNA fragments were amplified using indexed primers (Illumina) for 6 PCR cycles and purified with 0.9 x volume NEBNext Sample Purification Beads. Size distribution of the DNA libraries were checked and sequenced using NextSeq 500.