GSM9074385: human OCI-AML4, AAVS1 KO2, Day 3 post-editing; Homo sapiens; RNA-Seq
RNA was extract by following ChIRP-RNA isolation protocol. For each sample, 1×10⁷ MOLM13 or OCI-AML4 cells were fixed in 10 mL of freshly prepared 1% glutaraldehyde at room temperature for 10 minutes, then quenched with 125 mM glycine for 5 minutes. Fixed cells were washed in ice-cold DPBS, flash-frozen, and stored at -80 °C. Fixed cell pellets were thawed and resuspended in lysis buffer (1 mL per 10⁷ cells) supplemented with 1× PMSF, 1× protease inhibitor, and RNase inhibitor, then sonicated using a Covaris E220 ultrasonicator to obtain 100-500 bp chromatin fragments. The efficiency of fragmentation was verified by agarose gel electrophoresis after proteinase K digestion and DNA purification (Qiagen). For each ChIRP reaction, lysates were incubated with biotinylated probes (100 pmol per 1 mL lysate) in Hybridization Buffer at 37 °C for 4 hours with rotation. Pre-washed Dynabeads™ MyOne™ Streptavidin C1 beads (100 µL per 100 pmol probe) were added and incubated for an additional 30 min. Beads were washed five times at 37 °C using Wash Buffer to reduce nonspecific binding. For RNA isolation, beads were incubated in RNA PK Buffer with proteinase K at 50 °C for 45 min, followed by heat revere cross-linking at 95 °C for 10 min. RNA was extracted using TRIzol reagent and purified with the miRNeasy Mini Kit (Qiagen). Residual DNA was removed using the TURBO DNA-free™ Kit. 10 ng RNA was used for generating the sequencing library. SMART-Seq® Total RNA Pico Input Kit (Takara) was used to construction the library according to the manufacturer's instructions.