GSM9834862: MSK107Li PDO cells, shCtrl, 4sU label for 6hrs, chase for 0hrs, replicate 3; Homo sapiens; OTHER
Total RNA was subsequently isolated using a standard TRIzol-based extraction protocol. Purified RNA was subjected to chemical derivatization according to the SLAMseq Kinetics Kit – Catabolic Kinetics Module manufacturer's instructions. Briefly, 4sU-labeled RNA was alkylated with iodoacetamide (IAA) under the recommended reaction conditions to modify incorporated 4sU residues prior to library preparation. Alkylated RNA was purified and used as input for sequencing library construction. The SLAMseq chemistry enables reverse transcriptase to incorporate G opposite alkylated 4sU during cDNA synthesis, resulting in diagnostic T>C conversions in sequencing reads derived from newly labeled transcripts. Sequencing libraries were generated from total RNA using the QuantSeq 3′ mRNA-seq V2 Library Prep Kit FWD with Unique Dual Indices according to the manufacturer's instructions. This protocol uses oligo(dT)-primed first-strand synthesis, followed by RNA removal, random-primed second-strand synthesis, bead purification, and PCR amplification to generate strand-specific Illumina-compatible libraries enriched for the 3′ ends of polyadenylated transcripts. Unique dual indices were introduced during PCR amplification. Libraries were prepared from total RNA without prior poly(A) enrichment or rRNA depletion, consistent with the QuantSeq workflow.
GEO
GSM9834862_r1