GSM8742557: smallRNA_3end_globalseq_totalRNA_rep3; Homo sapiens; ncRNA-Seq
Small RNA (100-500 bp) was isolated with a 9% polyacrylamide/6M urea denaturing gel. Ribosomal (r)RNAs were depleted using RiboCOP rRNA depletion kit (Lexogen) per manufacturer's recommendations. A 3' adaptor containing a 15 or 16 bp randomer was ligated to RNAs. Nascent RNA was isolated with Click-it nascent RNA capture kit (Thermo Fisher). A fraction of material was prepared as total RNA and was not processed with nascent capture. A 5' adaptor was ligated following reverse transcription using a primer against the 3' adaptor. The cDNA was amplified in two stages of Polymerase Chain Reaction (PCR) using Q5 DNA polymerase (NEB). For the first PCR reaction, the cDNA library was amplified using 3' adaptor primer (AR17) and a primer complementary to the 5' adaptor (RC_3Tr3) for 8 cycles. The PCR product was purified by AMPure XP beads (Beckman Coulter) per manufacturer's recommendation. The second PCR reaction was performed using Illumina Trueseq D50X and D70X primers for 18 cycles.