GSM8844705: HEP3B, Control, IP, Rep 2; Homo sapiens; RIP-Seq
For cellular lysis, pellets were resuspended in 1 mL cold lysis buffer [50 mM Tris-HCl pH 7.4, 100 mM NaCl, 1% (vol/vol) Igepal CA-630, 0.1% (vol/vol) SDS and 0.5% (wt/vol) sodium deoxycholate, and 5.5 µL 200× protease inhibitor cocktail III], Lysates were sonicated in a Bioruptor sonicator (30s on/30s off at 4°C for 5 min). 5 µL Turbo DNase and 10 µL diluted RNase I (1:25 dilution in iced cold 1× PBS) were added to each lysate and then immediately incubated in a Thermomixer at 37°C and 1,200 rpm for 5 min. RNAse digestion was stopped by adding 11 µL murine RNase inhibitor. Lysis were cleared by centrifugation at 15000 ×g, 4°C for 15 min and 1 mL of each supernatant was collected for further use. NAT10 was immunoprecipitated with anti-NAT10 antibody (10 µg per sample, rabbit polyclonal) conjugated to M-280 sheep anti-rabbit IgG magnetic beads. To identify the NAT10 bound RNAs, a 3' Illumina adapter was ligated on-beads followed by separation of the complexes via SDS-PAGE. The NAT10-RNA complexes were excised out of the membranes, followed by proteinase K digestion and RNA isolation using the RNA Clean and Concentrator Kit (Zymo Research, R1018). Reverse transcription was performed using a specific primer aligning to the 3' Illumina adapter. Following cDNA purification, the other Illumina adapter, which contained a ten nucleotide UMI, was ligated to the 3' end of the cDNA. Final libraries were generated by performing PCR using Q5® High-Fidelity 2X Master Mix (New England Biolabs) and NEBNext® Multiplex Oligos for Illumina® (New England Biolabs) as primers. Libraries were gel purified and quality was verified on a TapeStation 4200 System (Agilent) before sequencing.  To process size-matched input (SMInput) samples, an aliquot of cell lysates was collected before the immunoprecipitation was carried out. Inputs were separated using SDS-PAGE and a region that matches NAT10's molecular weight was excised out of the membranes, followed by proteinase K digestion and RNA isolation using the RNA Clean and Concentrator Kit (Zymo Research, R1018). A 3' Illumina adapter was ligated followed reverse transcription using a specific primer aligning to the 3' Illumina adapter. Following cDNA purification, the other Illumina adapter, which contained a ten nucleotide UMI, was ligated to the 3' end of the cDNA. Final libraries were generated by performing PCR using Q5® High-Fidelity 2X Master Mix (New England Biolabs) and NEBNext® Multiplex Oligos for Illumina® (New England Biolabs) as primers. Libraries were gel purified, and quality was verified on a TapeStation 4200 System (Agilent) before sequencing.