title: GSM5901405: Dual-Seq_BatchB_EMB; Mycobacterium tuberculosis; Homo sapiens; RNA-Seq
library_construction_protocol: Dual RNA-Seq: To remove host RNA, Trizol samples were centrifuged and approximately 75% (~500 µL) of Trizol supernatant was transferred to a new tube. An additional 400 µL of Trizol and 150 µL of zirconia beads were added to the tubes containing the bacterial pellet and 25% of the remaining Trizol. These tubes were placed on a beat beater for two cycles of one minute each, with 2 minutes of rest on ice in between. After the bead beating, approximately 75% of host RNA in Trizol was added to be bacterial lysate tubes. Chloroform was added to Trizol tubes (200 µL of chloroform for 1 mL of Trizol) and tubes were mixed via shaking vigorously. Trizol:chloroform tubes were centrifuged for 15 minutes and the aqueous phase (~1 mL) was transferred to a fresh tube. 2 µL of glycoblue and 500uL of isopropanol were added to precipitate host and bacterial RNA. RNA was washed in 75% cold ethanol and RNA was eluted in 12 µL of NCFW. Cholesterol RNA-seq: Bacterial pellets in GTC buffer were washed with PBS + 0.05% Tween-80. Bacterial cells were lysed via 5mg/mL lysozyme treatment for 15 minutes at room temperature followed by hot Trizol bead beating for one cycle of 2 minutes. After bead beating, chloroform was added and the tubes were centrifuged to separate phases. The aqueous phase was then added to equal volume of pure ethanol. RNA was then purified using a Qiagen Rneasy Kit. Sequencing libraries were generated using the NEBNext Ultra™ II Directional RNA Library Prep Kit for Illumina (New England BioLabs). Libraries were sequenced on a HiSeq 4000 (Illumina) in multiple rounds until the desired sequencing depth for bacterial reads was reached.