title: GSM8098614: PGC1a  CUT&RUN replicate 1; Mus musculus; OTHER
library_construction_protocol: Preparation of nuclei suspension: The somatosensory cortex was dissected from P12 WT C57BL/6 mice in ice-cold buffer HB (0.25 M sucrose, 25 mM KCl, 5 mM MgCl2, 20 mM Tricine-KOH pH 7.8, 1 mM DTT, 0.15 mM spermine, 0.5 mM spermidine). Cortices were homogenized by a Dounce homogenizer in the presence of 0.3% IGEPAL. The homogenate was then filtered through a 40 µm strainer (Fisher Scientific #11597522), centrifuged at 4°C for 5 min at 500 g, and the pellet was washed twice with 1% BSA solution. The pelleted nuclei were resuspended in CUT&RUN Wash buffer (20mM Tris-HCl pH 7.5, 150mM NaCl, 0.5mM spermidine, 0.1% BSA), and the shape of the nuclei was verified using Tryptan Blue and a hemacytometer. All steps were performed on ice and using LoBind pipette tips. CUT&RUN: Nuclei were incubated with 2 μg of mouse anti-PGC-1⍺ antibody (Millipore #ST1202, three replicates) or 2 μg of mouse anti-ERR𝛾 antibody (R&D Systems #PP-H6812-00, one replicate) in CUT&RUN Wash buffer for 10min with rotation at 4°C. 2 μg of rabbit anti-mouse IgG secondary antibodies (Abcam #ab6709) were then added to the nuclei solution, which was left to rotate for 10min at 4°C. Nuclei were then incubated without washing step with pA-MNase (1.6 µg/µl) at 4 °C with rotation for an additional 10 min. Nuclei were then washed and resuspended with CUT&RUN wash buffer. DNA-tethered cleavage was induced by adding CaCl2 at a final concentration of 2 mM on ice water for 10 min. The reaction was stopped by adding Stop buffer (200 mM NaCl, 20 mM EDTA, 4 mM EGTA, 50 µg/ml RNAse A (Thermo Scientific #EN0531), 40 µg/ml glycogen (Thermo Scientific #R0561)) at a 1-to-1 ratio. The shape of the nuclei was again verified using Tryptan Blue and a hemacytometer. To release the DNA fragments of interest, nuclei were incubated at 37 °C for 20 min. Solubilized chromatin of interest was separated from nuclei by centrifugation at 16,000 g at 4°C for 5 min and then digested with SDS (0.1% final concentration) and Proteinase K (0.2 mg/ml final concentration) at 70°C for 10 min. Released DNA fragments were then extracted with phenol:chloroform:isoamylalcohol (25:24:1) and 13,000 rpm centrifugation for 15 min at room temperature. The aqueous upper layer was transferred to a fresh tube, and NaCl was added at a final concentration of 250 mM. An equal volume of isopropanol was further added, and the DNA was left to precipitate for 2 h at -80°C. Precipitated DNA was then pelleted by centrifugation (13,000rpm for 15 min at 4°C), washed with 70% ice-cold EtOH, and resuspended in distilled water. Libraries for sequencing were prepared using the NEBNext Ultra II library prep kit (New England Biolabs) following the protocol and thermocycling temperatures therein. Briefly, DNA ends from fragments harvested from the CUT&RUN experiment were repaired, 5' ends phosphorylated and 3' ends A tailed prior to ligation with NEBNext Adaptor, UNG was then used to linearize the ligated adapter. SPRIselect beads were used to deplete the unligated adaptor. Adaptor-ligated libraries were then barcoded by PCR following the manufacturer's recommended protocol. Libraries were amplified for between (11-13) cycles and purified using SPRIselect beads prior to quantitation using Qubit and HS DNA tapestation. Individually barcoded libraries were then pooled and sequenced on an Illumina NextSeq 2000 instrument.