Instrument: NextSeq 500
Strategy: OTHER
Source: TRANSCRIPTOMIC
Selection: other
Layout: SINGLE
Construction protocol: Cells were harvested by filtration and were scraped into liquid nitrogen. Lysis was conducted on a mixer mill (Qiagen) and lysate was treated with MNase and separated on a sucrose gradient to yield ribosome protected mRNA fragments. RNA was harvested by phenol chloroform extraction. See publication for details. RNA fragments were size-selected, dephosphorylated, and ligated to a linker RNA. RNA was then reverse transcribed using an oligo recognizing the ligated linker. cDNA was circularized and cDNA arising from rRNA was subtracted using custom oligos. Finally, libraries were PCR amplified and sequenced.