Instrument: Illumina HiSeq 2500
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: SINGLE
Construction protocol: RNA was isolated using a hot phenol method The isolated RNA was split into two equal groups and one group was treated with the enzyme RppH (NEB, M0356S) at 37°C for 2 hours to remove the outer two phosphate groups from the 5ʹ end of the non-processed RNA species (RppH+ sample). The other group is untreated (no incubation with RppH) and thus the non-processed RNA species retain a tri-phosphate at the 5ʹ end of these molecules (RppH- sample). Both groups are treated with NaIO4 for 1.5 hours at 4°C in the dark to inactivate the 3ʹ end of the RNA transcripts and reduce the production of side products during the subsequent ligation of amplification primers. The sequencing adapter with desired index barcode sequence is ligated to the 5ʹ end of RNA species with a single phosphate group. Note that RNA species with tri-phosphate groups at the 5ʹ end of the RNA are not substrates for ligation and thus will form a product with this sequencing adapter. The ligated RNA is purified using SPRI beads (Beckman, B23317), eluted in water, and quantitated using a NanoDrop 2000. Both the RppH+ and RppH- samples are subjected to rRNA depletion using the RiboZero kit (Epicentre, MRZB12424) following standard protocols and then reverse transcribed using a random primer attached to the second Illumina sequencing adapter following manufacturer's recommendations. PCR is used to amplify cDNA species in both the RppH- and RppH+ samples that contain both Illumina sequencing adapters using a KAPA Hi-Fi PCR-read mix (KAPA Biosystems KK2601) following manufacturer's protocol. The PCR products are cleaned using SPRI beads twice and resuspended in a final volume of 20 µL. The TSS-seq library is quantified using a 2100 Bioanalyzer (Agilent) and identifying fragments ~130 bp.