GSM8327270: N-term_MeDIP; Homo sapiens; MeDIP-Seq
1. MeDIP-Seq: After 72 h of transfection, cells over-expressing N-term & C-term truncated forms and F-len CGGBP1 were harvested for genomic DNA isolation using phenol-chloroform-isoamyl alcohol extraction method. Genomic DNA was sonicated to obtain 700-1000 bp fragments. 1µg fragment DNA was end repaired using NEBNext Ultra II End Repair/dA-Tailing Module (NEB #​​E7546). PCR adapters from Oxford Nanopore Technologies (ONT #SQK-PSK004) were ligated to end repaired DNA using NEB Blunt/TA Ligase Master Mix (NEB #M0367). 1x MeDIP master mix (10 mM Sodium Phosphate Buffer, 0.14 M NaCl and 0.05% TritonX-100) was added to adapter ligated DNA which was then denatured at 95℃ for 5 mins followed by snap chilling on ice. An antibody cocktail against 5-methylcytosine (EMD Millipore #MABE146, Sigma #SAB2702243 and Novus Biologicals #NBP2–42813) was added to snap-chilled DNA and was incubated at 4℃ overnight. Protein G Plus/Protein A Agarose beads were added to the mix followed by incubation at room temperature for 2 h. Then beads were collected by centrifugation in a separate tube and were washed thrice with 1x MeDIP master mix buffer and were subjected to Proteinase K (Sigma #P2308) digestion at 56℃ for 2 h. The slurry was centrifuged and immunoprecipitated DNA in the supernatant was collected into new tubes. This DNA was subjected to 18 cycles of PCR using whole genome primers (ONT #SQK-PSK004) and LongAmp Hot Start Taq 2X Master Mix (NEB #M0533). 2. ChIP-Seq: The N-term, C-term, and FL-CGGBP1 were over-expressed in HEK293T-shB-KD, HEK293T-shA-KD, and HEK293T-CT cells, respectively. Cells were harvested for ChIP after being transfected for 72 hours. The cells were harvested using a scrapper after being crosslinked with 4% formaldehyde for 10 minutes at 37°C. They were then quenched with glycine (125 mM). Thermo Fisher Scientific #87785's 1x Halt Protease inhibitor cocktail, EDTA-free, was added to SDS lysis buffer (50 mM Tris pH 8.0, 10 mM EDTA, 1% SDS) and the mixture was lysed for 30 minutes on ice with periodic flick-mixing. After that, DNA was sonicated for 20 cycles of 30 seconds ON and 30 seconds OFF to create DNA fragments that ranged in length from 0.15 to 0.35 kb. The fragmented DNA was diluted in ChIP-dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl pH 8.1, 167 mM NaCl) after taking out 10% DNAseparately as input. In order to pre-clear chromatin, Protein G Plus/Protein A Agarose beads (Merck Millipore #IP05) were incubated for 4hours at 4°C. After that, the DNA was incubated for an overnight period at 4°C with gentle tumbling with an antibody cocktail targeted against the N-term(Invitrogen #PA5-57317, Abcam #ab176814, Abclonal #AE005 directed towardsthe FLAG-tagged N-term). Protein G Plus/Protein A Agarose beads were then added to collect the antibodies, and the mixture was tumble-incubated for an hour at room temperature. After that, the beads were pelleted and washed with the following four buffers: LiCl IP wash buffer (0.25 M LiCl, 1% IGEPAL (NP40), 1% sodium deoxycholate, 1 mM EDTA, and 10 mM Tris–HCl); low-salt IP wash buffer (0.1% SDS, 1% Triton X–100, 2 mM EDTA, 20 mM Tris–HCl and 150 mM NaCl); and two washes of TE buffer (10 mM Tris–HCl and 1 mM EDTA). An elution buffer (1% SDS and 0.1 M NaHCO3) was used to elute the immunoprecipitated DNA in a 500ul total volume. 200 mM NaCl was added to eluted DNAand incubatedfor 8 hours at 65°C forreverse-crosslinking. Next, for one hour at 45°C, Proteinase K (Sigma #P2308) was added to the DNA along with 10 mM EDTA, 0.1 M Tris-HCl pH 6.8, and 0.1 mg/ml for protein digestion. After adding 10% v/v 3M sodium acetate at pH 5.2, ethanol precipitation was used to purify the ChIP DNA. 1. MeDIP-Seq: Library preparation was done using NEBNext® UltraTM II DNA Library Prep Kit for Illumina® (NEB #E7645, #E7103) as per manufacturer's instruction. DNA was quantified using Qubit and quality assessment was done using TapeStation. DNA was sequenced on Illumina HiSeq 2500 platform. 2. ChIP-Seq: Library preparation was done using NEXTFlex Rapid DNA Sequencing Bundle (NEXTflex, #5188-12) as per manufacturer's instruction. DNA was quantified using Qubit and quality assessment was done using TapeStation. DNA was sequenced on Illumina NovaSeq 6000 platform. MeDIP-Seq & ChIP-Seq