GSM7598040: meRIP seq Input of MOLM13:DMSO-treated rep2; Homo sapiens; OTHER
For Cut&Tag, 5 × 104 MOLM13 cells were sorted and washed once in 0.5 mL wash buffer (20 mM Hepes (pH 7.5); 150 mM NaCl; 0.5 mM spermidine; and 1 × protease inhibitors) and were incubated with 10 μL pre-washed concanavalin A–coated magnetic beads in a 1.5 mL microtube at 25 °C for 10 min. Cells were resuspended in 80 μL ice-cold antibody incubation buffer with 1 μg RBM15B antibody per sample (22249-1-AP, proteintech) and then rotated end-over-end at 4 °C overnight. The cells were resuspended in 100 μL of dig-wash buffer [20 mM Hepes (pH 7.5); 150 mM NaCl; 0.5 mM spermidine; 1 × protease inhibitors; 0.025% digitonin) with rabbit anti-mouse IgG (1:100 dilution, Ab6709; ThermoFisher) and rotated end-over-end at 25 °C for 1 h. After washing, home-made pA-Tn5 adapter complexes in dig-300 buffer (20 mM Hepes pH 7.5; 300 mM NaCl; 0.5 mM spermidine; 1 × protease inhibitors; 0.01% digitonin) were added to the samples at a final concentration of 1:100 and then rotated end-over-end at 25 °C for 1 h. After washing, the cells were resuspended in 300 μL fragmentation buffer (dig-300 buffer with 10 mM MgCl2) and incubated at 37 °C for 1 h. After extraction with phenol–chloroform and ethanol precipitation, the libraries were amplified by PCR.  For m6A-RIP, Total RNA was extracted using TRIzol™ Reagent (Invitrogen, 15596018). The concentration of total RNA was measured by Qubit RNA HS assay kit (Invitrogen, Q32852). 10μg total RNA was fragmented into 100-200 nt RNA fragments using 10X RNA Fragmentation Buffer (100 mM Tris-HCl, 100 mM ZnCl2 in nuclease-free H2O). The fragmented RNA was incubated with anti-m6A monoclonal antibody (abcam,ab208577) for 3 h at 4℃ and then with protein A/G magnetic beads (Invitrogen, 8880210002D/10004D) at 4 ℃ for an additional 2 h to obtain immunoprecipitated RNA fragments. The m6A-enriched RNA was purified using TRIzol™ Reagent (Invitrogen, 15596018). For Cut&Tag sequencing, the barcoded libraries were pooled and sequenced on the Illumina HiSeq X™ Ten platform to generate 150 bp paired-end reads. For m6A-RIP sequencing, the library was prepared by smart-seq method. Both the input samples without IP and the m6A IP samples were subjected to 150-bp, paired-end sequencing on an Illumina NovaSeq 6000 sequencer.