NaP-TRAP utr5 fish pA vs SV40 - ntrap 2h pulldown pA B1
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.