PCR amplification was done with Platinum Taq DNA polymerase (Thermo Fisher Scientific 10966018) using 0.5 μM 3′ RT primer and 5′ SHORT PCR primer in a 100-μL reaction volume. After eight cycles from PCR (10 sec at 94°C, 30 sec at 60°C, and 15 sec at 72°C), the samples were concentrated and purified using DNA Clean & Concentrator-5 (Zymo Research, Catalog no. D4013). Products of a size between 75 and 100 bp were isolated using a 3% agarose PippinPrep cassette (Sage Science CSD3010) on a BluePippin device.
The second PCR was done with the purified and size-selected samples using Platinum Taq DNA polymerase together with 0.5 μM 5′ long PCR primer and 3′ RNA index primer (barcoded primer) in 100-μL reaction volume for 15 cycles. The PCRs were again purified, and size-selected for a range between 147 and 173 bp. The resulting library was sequenced on a HiSeq 3000 system using a single-end 50 cycle protocol. Analysis was performed as described previously using PARalyzer (version 1.5; Corcoran et al. 2011 (link)) built into the PARpipe (Corcoran et al. 2011 (link)) pipeline mapping the reads to human genome hg19. Pooled version of reads was mapped on human genome hg38.
The second PCR was done with the purified and size-selected samples using Platinum Taq DNA polymerase together with 0.5 μM 5′ long PCR primer and 3′ RNA index primer (barcoded primer) in 100-μL reaction volume for 15 cycles. The PCRs were again purified, and size-selected for a range between 147 and 173 bp. The resulting library was sequenced on a HiSeq 3000 system using a single-end 50 cycle protocol. Analysis was performed as described previously using PARalyzer (version 1.5; Corcoran et al. 2011 (link)) built into the PARpipe (Corcoran et al. 2011 (link)) pipeline mapping the reads to human genome hg19. Pooled version of reads was mapped on human genome hg38.
