GSM8153855: CC,ChIP,REST,rep3; Homo sapiens; ChIP-Seq
ChIP–qPCR analysis ChIP-seq sample preparation was carried out as previously described49. Briefly, SHSY-5Y cells WT and SIRT6 KO, or WT and SIRT KO transfected with Flag-REST, K494A, K494M, K494Q were cultured up to 80% of confluence. In the case of ChIP negative controls cells were transfected with an empty vector (CMV-Flag). Cells were cross-linked with 1% formaldehyde for 10 minutes and blocked with 0.125M Glycine for 5 minutes. Cross-linked cells were scrapped on RIPA buffer (150mM NaCl, 1% NP-40, 0.5% Sodium deoxycholate, 0.1% SDS, 50mM Tris pH 8, 5mM EDTA, 0.5mM PMSF, 50mM NaF/0.2mM Sodium orthovanadate, 5uM trichostatin A), and chromatin was sonicated with a needle sonicator (VibraCell VCX130, Sonics, CT, USA) during 25 minutes, 30/30 seconds on/off cycles at 40% of amplitude. One milligram of the sonicated chromatin was incubated overnight with previously blocked SureBeads™ Protein G Magnetic Beads or Anti-FLAG® M2 Magnetic Beads. The beads were then washed 4 times with RIPA buffer, 4 times with LiCl buffer (500mM LiCl, 100mM Tris-HCl pH 8.5, 1% NP-40, 1% sodium deoxycholate), and 2 times with TE buffer (10mM Tris-HCl pH 8, 1mM EDTA). Chromatin was eluted from beads using elution buffer (70mM Tris-HCl pH 8, 1mM EDTA, 1.5% SDS) and cross-linking was reversed by adding 200mM NaCl, 1U of Proteinase K, and incubating the eluted chromatin at 65C for 5 hours. The obtained DNA was further purified using the Nucleo Spin Gel and PCR Clean-Up kit. For qPCR analysis of specific REST binding regions in the genome, we used the set of primers listed in the key resources table. qPCR was performed according to the method described. We used 1uL of Input and ChIP samples per qPCR reaction. ChIP DNA samples were quantified using Qubit 2.0 Fluorometer (Life Technologies, Carlsbad, CA, USA) and the DNA integrity was checked with 4200 TapeStation (Agilent Technologies, Palo Alto, CA, USA). ChIP-Seq library preparation and sequencing reactions were conducted at GENEWIZ, Inc/Azenta US, Inc. (South Plainfield, NJ, USA). NEB Next Ultra II DNA Library Preparation kit was used following the manufacturer's recommendations (Illumina, San Diego, CA, USA). Briefly, the ChIP DNA was end repaired and adapters were ligated after adenylation of the 3'ends. Adapter-ligated DNA was size selected, followed by clean up, and limited cycle PCR enrichment. The ChIP library was validated using Agilent TapeStation and quantified using Qubit 2.0 Fluorometer as well as real time PCR (Applied Biosystems, Carlsbad, CA, USA). The sequencing libraries were multiplexed and clustered on one lane of a flowcell. After clustering, the flowcell was loaded on the Illumina Novaseq instrument according to manufacturer's instructions (Illumina, San Diego, CA, USA). Sequencing was performed using a 2x150 Paired End (PE) configuration. Image analysis and base calling were conducted by the Novaseq Control Software (NCS). Raw sequence data (.bcl files) generated from Illumina instrument was converted into fastq files and demultiplexed using Illumina's bcl2fastq 2.17 software. One mis-match was allowed for index sequence identification.