Instrument: Illumina NovaSeq 6000
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: PAIRED
Construction protocol: Total RNA was isolated using the Qiagen Rneasy Mini Kit (Catalog Number 74104) with on-column Dnase treatment (Catalog Number 79254) according to manufacturer protocol. Tn5Prime libraries were constructed essentially as described by Cole and colleagues (NAR, 2018). Briefly, RNA was reverse-transcribed by SMARTScribe RT in the presence of an oligo-dT primer and template-switching oligo. The reaction was treated with RNase and Exonuclease. The resulting DNA was subjected to an indexing PCR, tagmentation by purified Tn5 enzyme pre-loaded with Tn5ME-B/R adapters, and another round of PCR. Size selection to 400-1000bp was performed on a 0.8% low-melt agarose TAE gel. DNA was purified using the Qiagen Gel Extraction Kit (Catalog Number 28704). Libraries were sequenced on Illumina HiSeq or NovaSeq sequencers. Read 1 (corresponding to 5' end of mRNAs) was used for subsequent analyses.