title: NaP-TRAP UTR5 library HEK293T cells - pulldown-raw
design: Reverse transcription primers (4 M total) targeting the N-terminus of 3xFLAG GFP were added to purified input and pulldown RNAs (5-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-CTAC-10N UMI-TAAC-6 nt sample barcode-GCGTCATGGTCTTTGTAGTCCTC-3). These primers contained a 6 nucleotide sample barcode and a 10 nucleotide Unique Molecular Identifier (UMI) to allow for demultiplexing and read deduplication, as well as a 3 I7 Illumina adaptor sequence. To increase library complexity the primer pairs were staggered by 1 nt (e.g., ntrap_RT_b1.1, ntrap_RT_b1.2). Reverse transcription was performed using the Superscript III kit (Invitrogen #18080044) in accordance with manufacturers instructions. Reverse transcription reactions were performed at 55C. cDNA from replicates were pooled and purified by adding AMPure XP Reagent (Beckman Coulter #A63881) at 1.8x of the original sample volume. Illumina I5 and I7 forward and reverse primers containing a 10-nucleotide index (see below) were utilized to amplify cDNA libraries via PCR (Kappa Polymerase Master Mix). To reduce the number of PCR duplicates 12-18 cycles were utilized. Amplicons were purified by adding AMPure XP Reagent (Beckman Coulter #A63881) at 0.9x the volume of the PCR reaction. Libraries were sequenced on Illumina NovaSeq 6000 platform. Paired-end reads were trimmed and demultiplexed using ReadKnead (https://github.com/vejnar/ReadKnead). Barcodes identifying replicates and UMIs were extracted from read two.