High sensitivity chip kit

ChIP assay was performed with High-Sensitivity ChIP Kit (Abcam, Cambridge, UK) following manufacturer's instructions. Briefly, cells were lysed and the pellet containing chromatin was resuspended with 500 μl ChIP Buffer. The chromatin was then extracted by sonication shearing. The ChIP reaction mixtures were incubated at 4°C overnight, and the DNA was released and purified for the following PCR test.

The subcortical WM (SCWM) of WT mice from P12-P15 (reared under Nx and Hx conditions) was dissected and snap frozen. Chromatin was sonicated and immunoprecipitation with anti-Sirt2 antibody (Santa Cruz, sc211033) or IgG negative control was performed using the High Sensitivity ChIP kit from Abcam (Abcam 185913). DNA samples were sent to the Penn State Hershey Genome Sciences and Bioinformatics Facility and Penn State College of Medicine. Libraries were prepared using the NebNext kit for input of <100 ng DNA, according to the manufacturer’s protocol. 50 base pair, paired-end reads were obtained from the NovaSeq 6000 System (Illumina). Reads were mapped to the mouse reference genome (Genome Reference Consortium Mouse Build 38—mm10) using bowtie2 (version 2.4.4)^{82 (link)}, and mitochondrial chromosomes were removed using Samtools (version 1.7)^{83 (link)}. Duplicates were marked with Picard (version 2.26.10) and removed with Samtools. Binding peaks were identified using Macs2 (version 2.2.7.1)^{84 (link)} using an adjusted p value <0.1 as threshold. Sirt2 ChIP-seq data were compared with the RNA-seq data previously published^{39 (link)}.

ChIP-qPCR was performed by using the High-Sensitivity ChIP Kit (Abcam, #ab185913) according to the manufacturer’s protocol. In brief, Tsc2 KO MEFs or 105K cells were crosslinked by 1% formaldehyde and chromatin was extracted and sheared by a M220 Focused-ultrasonicator (Covaris). Samples were immunoprecipitated with anti-YY2 antibody (Santa Cruz Cat#sc-377008). The primer sequences used for ChIP-qPCR are listed in the Supplementary Table 3. The results were from 3 biological samples followed by normalization to input signals and showed as mean ± SD.

BEAS-2B Cells were washed twice with PBS and cross-linking was performed in 9 mL culture medium containing 1% formaldehyde at room temperature for 15 min. The reaction was halted by adding glycine to the final concentration of 125 mM. Chromatin extraction was performed with the High Sensitivity ChIP Kit (Abcam) as per the manufacturer’s instructions. A total of 2 μg chromatin was used for the ChIP with anti-HIF-1α antibody overnight at 4 °C. Following cross-link reversal and DNA purification, 1 μL of the eluted DNA was used for quantitative polymerase chain reaction with target region primers: 5'-GGATCGACCTGACTGAACCT-3' and 5'-CCTGGGATCCGAACGGATCT-3'.

Cells were treated with decitabine and azacitidine or vehicle in 6-well plates for 72 h. The ChIP assay was performed using High Sensitivity ChIP Kit (Abcam) according to the manufacturer’s instructions. After washing, protein and DNA complexes in cells were crossed linked in 1% formaldehyde culture medium at room temperature for 10 min. DNA in the nuclear fraction was sheared by sonication and the supernatant containing the protein-DNA complex were incubated with an antibody (Abcam) to acetyl-histone H3 (1 µg) in each well for 1 h. Following decrosslinking and proteinase K treatment, the antibody-precipitated DNA was purified. The DNA was amplified using PCR with T100 Thermal Cycler (Bio-Rad), and PCR product was analyzed by 4% agarose gel. The primers and PCR conditions to amplify the CDKN2A and CDKN2B promoters were described previously^{42 (link)}.

ZIP13K2-derived EN cells (2x10⁶ / IP) were harvested, cross-linked, washed, lysed, and sonicated as described previously (s. SOX17 ChIP sequencing). ChIP for H3K9me3 was performed in triplicates utilizing the High-Sensitivity ChIP Kit (abcam, ab185913) in combination with the ChIP-grade H3K9me3 antibody (ab8898, abcam) according to the manufacturer’s instructions with slight modifications. Instead of DNA column purification, phenol:chloroform extraction followed by precipitation was performed to isolate DNA (s. SOX17 ChIP sequencing). Precipitated DNA was dissolved in 200 µl H₂O.
qPCR reactions were set up utilizing the 2 x PowerUp SYBR Green Master Mix (Thermo Fisher Scientific, A25777) containing final 250 nM forward /reverse primer (s. Supplementary file 3). All samples have been measured in technical triplicates using 4 µl diluted input or IP sample from above /reaction /replicate. qPCRs were set-up on 96-well plates (Thermo Fisher Scientific, N8010560), spun down for 1 min at 2500 x g, RT and ran on a StepOnePlus 96-well Real-Time PCR System (Thermo Fisher Scientific).