title: GSM5421575: rz_frg_5m-chpb1; Escherichia coli; RNA-Seq
library_construction_protocol: RNA was extracted using Trizol (Invitrogen) and Direct-zol RNA MiniPrep kit (Zymo). See publication for complete details. For samples with rRNA depletion, rRNA was removed by hybridization to custom biotinylated oligos. Next, if samples were fragmented, RNA was mixed with reverse transcription buffer and heated to 95°C for 3 minutes before placing on ice. All samples were then reverse transcribed using reverse transcription primers with a 5'-overhang with a PCR amplification handle and 1 of 24 inline barcodes. As cDNA now contained a barcode, samples were pooled and those with a tRNA size selection step were loaded onto a denaturing TBE urea gel and the tRNA region of the gel was extracted for cDNA purification. For all other samples, pools were cleaned up with a modified AMPure XP bead protocol to remove leftover primers. Next, all samples were subjected to a second round of reverse transcription with a template switching oligo to add a second PCR amplification handle to the 3'-end of the cDNA. After a bead cleanup, samples were PCR amplified with primers adding the i5 and i7 handles for Illumina sequencing. PCR reactions were size selected with a 2-sided bead cut and sequenced using a custom read 1 primer on a NovaSeq instrument. The combination of the i7 index and the inline read 2 barcode were used to separate out biological samples into their own fastq files using cutadapt.