title: GSM5418073: D28 Mtb infected_T863 treated animal6 [SD17]; Mus musculus; RNA-Seq
library_construction_protocol: Total lung RNA extracted using RNAzol reagent, RNA samples were purified using RNeasy mini kit from Qiagen. Purified RNA was further treated with turbo Dnase and later 1 μg of total RNA was used for the construction of sequencing libraries. A modified NEBNext 594 RNA Ultra II directional protocol was used to prepare the libraries for total RNA sequencing. 595 The first step involved the removal of ribosomal RNA of cytoplasmic and mitochondrial origin 596 using biotinylated, target-specific oligos combined with rRNA removal beads. Following 597 purification, the RNA was fragmented using divalent cations under elevated temperature. 598 Next, the cDNA was synthesized using Reverse transcriptase and random hexamers in a 599 first strand synthesis reaction. Subsequently, the cDNA was converted to double stranded 600 cDNA where Uracil was added instead of Thymine. The strand specificity was preserved by 601 a USER enzyme based digestion of second strand thereby leaving one functional strand 602 which maps to the DNA strand which it was transcribed from. The USER digested single 603 strand molecules were enriched and indexed in a limited cycle PCR followed by AMPure 604 bead purification to create final cDNA library for sequencing.