title: GSM5099776: RNA GAS strain 854 [WT_A]; Streptococcus pyogenes; RNA-Seq
library_construction_protocol: Cell pellets were mechanically disrupted by bead beating in Trizol reagent. Chloroform was added and RNA was extracted from the aqueous phase via Direct-Zol RNA Miniprep Kit (Zymo Research) Illumina cDNA libraries were generated using a modified version of the RNAtag-Seq protocol. 500 ng - 1 µg total RNA was fragmented, depleted of genomic DNA, dephosphorylated and ligated to DNA adapters carrying 5'-AN8-3' barcodes of known sequence with a 5' phosphate and a 3' blocking group. Barcoded RNAs were pooled and depleted of rRNA using the RiboZero rRNA depletion kit (EpiCentre). Pools of barcoded RNAs were converted to Illumina cDNA libraries in 2 main steps: (i) reverse transcription of the RNA using a primer designed to the constant region of the barcode adaptor with addition of an adapter to the 3' end of the cDNA by template switching using SMARTScribe (Clonetech); (ii) PCR amplification using primers whose 5' ends target the constant region of the 3' or 5' adaptors and whose 3' ends contain the full Illumina P5 or P7 sequences.