title: GSM5047310: D39 Δrr09 mutant strain_replicate 3 [6764]; Streptococcus pneumoniae D39; RNA-Seq
library_construction_protocol: The pellets were processed for total RNA extraction using acid phenol-chloroform and DNase treatment to remove genomic DNA. The products were purified using the RNA Clean-Up and Concentration kit (NORGEN BIOTEK CORP), the quality of the RNA was determined by agarose gel electrophoresis and the amount was quantified using a Nanodrop ND-1000 (PeqLab) RNA samples were fragmented, depleted of genomic DNA and dephosphorylated prior to their ligation to 5' phosphorylated barcoded adapters, Barcodes RNAs were pooled, depleted of rRNA using RiboZero and converted to Illumina cDNA libraries in 2 steps. First, the RNA was reverse transcribed using a primer targeting the ligated adapter. During cDNA synthesis, a second adapter was added to the 3' end of cDNAs through template switching. Second, cDNAs were PCR amplified with primers targeting the ligated adapters and carrying the full sequence of the Illumina sequencing adapters.