GSM7815494: Senescent NP cells treated with DMSO #1 for p65 CUT&Tag; Homo sapiens; OTHER
DNA were isolated with p65 (CST, #8242) antibodies. Briefly, collected nucleus pulposus (NP) cells were immobilized on Concanavalin A-coated magnetic beads (ConA), permeabilized with digitonin and incubated with p65 (CST, #8242)  antibody (1st Ab). The following day, ConA-nuclei-1st Ab were spun, and the supernatant was removed, nuclei were resuspended in 100 Î¼l of Wash Buffer 150 and incubated with a secondary antibody (2nd Ab) diluted 1:100 in Wash Buffer 150 for 1 h at room temperature. After removal of the 2nd Ab, nuclei were washed with Wash Buffer 150 and incubated with the protein AG-conjugated-Transposase (pAG-Tn5) preloaded with sequencing adapters, diluted 1:20 in Wash Buffer 300 for 1 h at room temperature. Nuclei were then washed twice in Wash Buffer 300 and tagmentation reaction was performed by addition of 10 mM MgCl2 following incubation at 37 Â°C for 1 h. After a wash with TAPS buffer (10 mM TAPS), tagmented DNA was released by addition of 5 Î¼l 0.1% SDS release buffer following incubation at 58 Â°C for 1 h. Finally, 15 Î¼l  of 0.67% Triton-X-100 were added to neutralize SDS and allow PCR for library preparation. Libraries were prepared according to instructions of Illumina HiSeq X ten accompanying the Ovation RNA-Seq System V2 (M01206 v9). Briefly, RNA was elutated according to the manuscription user guide. Then RNA was fragmented using fragment buffer. cDNA was synthed using dNTPs, DNA polymeraseI and RNaseH. Then cDNA was purified using AMPure XP beads. PCR Was used to amplify with Illumina primers for 15 cycles and library fragments of ~200 bp (insert plus adaptor and PCR primer sequences) were band isolated from an agarose gel. The purified RNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on the Illumina HiSeq X ten following the manufacturer's protocols. 2 Î¼l of 10 Î¼M i5 universal adapter primer and 2 Î¼l of 10 Î¼M i7 uniquely barcoded adapter primers were added to released, tagmented DNA, followed by addition of 25 Î¼l of 2X Master Mix. PCR was performed as follows: Cycle 1: 72 Â°C for 3 min (gap filling), Cycle 2: 95 Â°C for 3 min, Cycle 3: 98 Â°C for 10 s, Cycle 4: 60 Â°C for 5 s. 72Â°C for 1 min and hold at 4 Â°C. For p65 CUT&Tag, repeat Cycles 3-4 15 times. Amplified DNA was then recovered through size exclusion purification by addition of VAHTS DNA Clean Beads, followed by two washes in 80% ethanol and elution in 22 Î¼l of 10 mM Tris-HCl (pH8). Quantification and average fragment size of libraries was performed via the 4200 TapeStation system (Agilent), using the D5000 high sensitivity kit (Agilent 5067-5592/5067-5593). 450bp of average library fragment size was selected for library quantification. A 5 nM pool of the libraries was then run with the NovaSeq instrument and sequenced in paired end (PE), generating 2x150 bp reads.