GSM8488590: PANC-1 cell, shControl2; Homo sapiens; OTHER
The PANC-1 cells were divided in two groups: 1.PANC-1 transfected with siRNA Ctrl; 2.PANC-1 transfected with siRNA that knockdown the expression of NAT10. 30 μg total RNA was used for mRNA capture using KAPA mRNA Capture Kit(KK8441) following the manufacturer's instruction. Take a small amount of captured RNA as “Con”, and the remaining RNA was treated with 100 mM NaBH4 at 55℃ in the dark for 30 minutes. During the incubation time, tube lids were opened every 10 min to release pressure from NaBH4-induced bubbling. Reactions were adjusted to 200 μl with H2O, precipitated with 0.3M sodium acetate [pH 5.5], 15 mg/ml linear acrylamide (carrier) and 2.5X ethanol, briefly dried on Speedvac, resuspended in 12 μl H2O, and quantified by Qubit4.0 with Qubit™ RNA HS Assay Kit (Life Technologies, Q33224). Total RNA was extracted from shCtrl or shNAT10 PANC-1 cells using TRIzol Reagent (Invitrogen, cat. NO 15596026) following the methods by Chomczynski et al (DOI:10.1006/abio.1987.9999). 30 μg total RNA was used for mRNA capture using KAPA mRNA Capture Kit(KK8441) following the manufacturer's instruction. Take a small amount of captured RNA as “Con”, and the remaining RNA was treated with 100 mM NaBH4 at 55℃ in the dark for 30 minutes. During the incubation time, tube lids were opened every 10 min to release pressure from NaBH4-induced bubbling. Reactions were adjusted to 200 μl with H2O, precipitated with 0.3M sodium acetate [pH 5.5], 15 mg/ml linear acrylamide (carrier) and 2.5X ethanol, briefly dried on Speedvac, resuspended in 12 μl H2O, and quantified by Qubit4.0 with Qubit™ RNA HS Assay Kit (Life Technologies, Q33224). KC Digital Stranded mRNA Library Prep Kit for Illumina（DR085-02）was used for library construction. The kit eliminates duplication bias in PCR and sequencing steps, by using unique molecular identifier (UMI) of 8 random bases to label the pre-amplified cDNA molecules. The library products corresponding to 200-500 bps were enriched, quantified and finally sequenced on Novaseq 6000 sequencer (Illumina) with PE150 model.