Ab76307

After the cell confluence reached about 70%–80%, the cells were fixed with 1% formaldehyde at ambient temperature for 10 min. After cross-linking, they were randomly broken by ultrasonication, with 15 cycles of 10 s of ultrasound at 10 s intervals. The cells were centrifuged at 13,000 rpm at 4°C. The supernatant was incubated with antibodies: rabbit anti-immunoglobulin G (IgG) (ab109489, 1:100, Abcam, Cambridge, UK), anti-H3K9Ac (Millipore 07-352, Billerica, MA, USA), and anti-H3K56Ac (ab76307, Abcam, Cambridge, UK) overnight at 4°C. The endogenous DNA-protein complex was precipitated with protein agarose/Sepharose, the supernatant was discarded after a short centrifugation, and the non-specific complex was washed. After de-crosslinking at 65°C overnight, DNA fragments were harvested by phenol/chloroform extraction for measuring enrichment of H3K9Ac and H3K56Ac in Lin28b promoter fragment-specific primers.

Chromatin for native ChIP of histone modifications was digested with Micrococcal nuclease (MNase) and immunoprecipitated as described^{69 (link)}. For SIRT6, 40 × 10⁶cells (L-C-B and SIRT6.2–7) were crosslinked with 0.4% PFA for 10 min at RT. ChIP was performed as described^{70 (link)}. Immunoprecipitations were performed with 10 µg of specific antibodies anti-SIRT6 (Abcam, Ab62739), anti-H3K4me1 (Abcam, ab8895), anti-H3K4me3 (Abcam, ab1012), anti-H3K9ac (Abcam, Ab10812), anti-H3K27ac (Abcam, ab4729), and anti-H3K56ac (Abcam, ab76307). Sequencing libraries were generated and barcoded for multiplexing as described^{69 (link)}. Libraries were sequenced on NextSeq500 (50–75 bp single-end reads). Reads were trimmed for Illumina adapter sequences using in-house scripts and aligned to the GRCh37/hg19 using Bowtie (version 0.12.7) with parameters -l 65 -n 2 -S -best -k 1 -m 20. SIRT6 significant peaks were identified using MACS2 (version 2.1.1.2) and matching Input control was used to call peaks. Coverage tracks were generated from BAM files using deepTools (version 2.4.1) bamCoverage with parameters -normalizeUsingRPKM -binsize 10. Differential ChIP-seq-binding profiles for the histone marks H3K9ac and H3K56ac between L-C-B and SIRT6.2–7 cells were found by using MACS2 bdgdiff (parameters: -l 100 –g 100 or –l 250 –g 250 for H3K9ac and H3K56ac, respectively).

Tissue sample were fixed overnight in 4% paraformaldehyde and further embedded in paraffin. All stainings were performed on 4 μm sections. Hematoxylin and Eosin (H&E) stainings were carried out according to the standard procedure with an ASS1 staining unit (Pathisto). Fluorescence stainings were performed with the DyLight System (ThermoScientific) or the Tyramide Signal Amplification Kit (PerkinElmer), according to the manufacturer’s instruction. The slides were counterstained with DAPI and mounted in ProLong Gold (Invitrogen). PKCδ stainings were performed on 12 μm cryosections, which were air-dried, washed in PBS, blocked with 1% bovine serum albumin (BSA), 0.1% Triton X-100 in PBS and incubated in the primary PKCδ antibody (610397, BD) overnight followed by washing and incubation with the secondary antibody (DyLight 488; ThermoScientific).
Ki67 IHC detection was done with the IDetect Super Stain System HRP (ID laboratories), visualized with 3-amino-9-ethylcarbazole (ID laboratories) and counterstained with Hematoxylin.
Primary antibodies used for IHC: Ki67 (NovoCastra), HDAC1 (ab7028, Abcam), HDAC2 (3F3), NeuN (MAB377C3, Chemicon), GFAP (3670, Cell Signaling), H3K56ac (ab76307, Abcam), γH2AX (phospho S139) (ab2893, Abcam), cleaved caspase-3 (9661, Cell Signaling), TuJ1 (ab14545, Abcam).