title: GSM5100074: Mtb transcriptome - Hspx Low_1; Mus musculus; RNA-Seq
library_construction_protocol: Single cell suspensions from mice infected with hspx'::GFP/smyc'::mCherry (n=3, 3 batches, total of 9 mice) have been prepared as described previously (Pisu et al, 2020). The two different populations of infected host-cells (hspx-high and hspx-low) have been sorted (Figure S1A) and collected directly in Trizol, as previously described.(Pisu et al., 2020a) Dual RNA extraction has been performed as we previously reported (Pisu et al., 2020a). rRNA depletion has been performed using a 1:1 mix of depletion solutions from the Ribo-Zero H/M/R and Ribo-Zero Gram+ kits (Illumina) and a modified protocol previously described. (Pisu et al., 2020b) The rRNA-depleted samples were purified by ethanol precipitation. Sequencing libraries were generated using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (New England Biolabs). Libraries were sequenced on a Novaseq 6000 S4 (Illumina) in multiple rounds until the desired sequencing depth for bacterial reads was reached (target 1M 2x150nt PE reads).