title: GSM7078041: Atg5fl/fl LysMcre+/- macrophages, infected with M. tuberculosis, biol rep 2; Mus musculus; RNA-Seq
library_construction_protocol: Cells were resuspended in 1 ml of Trizol after 48 h of infection. 200 microliters of chloroform were added before centrifugation at 15,000 g for 10 min at 4°C. An equal volume of 70% ethanol was added to the aqueous layer, and RNA was extracted using silica spin columns (Invitrogen PureLink RNA Mini Kit). Purified RNA samples were treated with DNase (Purelink) and EDTA. Gene expression profiling was carried out using a 3' Tag-RNA-Seq protocol. Barcoded sequencing libraries were each prepared from 200 ng total RNA sample using the QuantSeq 3' mRNA-Seq Library Prep FWD kit for multiplexed sequencing, according to the recommendations of the manufacturer (Lexogen, Vienna, Austria). The fragment size distribution of the libraries was verified via micro-capillary gel electrophoresis on a LabChip GX system (PerkinElmer, Waltham, MA). The libraries were quantified by fluorometry on a Qubit fluorometer (LifeTechnologies, Carlsbad, CA), and pooled in equimolar ratios. The library pool was Exonuclease VII treated (NEB, Ipswich, MA), SPRI-bead purified with KapaPure beads (Kapa Biosystems/Roche, Basel, Switzerland), quantified via qPCR with a Kapa Library Quantification kit (Kapa Biosystems) on a QuantStudio5 RT-PCR system (Applied Biosystems, Foster City, CA). Libraries were sequenced on a NextSeq500 sequencer (Illumina, San Diego, CA) with single-end 85 bp reads. 3' Tag-RNA-Seq (see: https://dnatech.genomecenter.ucdavis.edu/tag-seq-gene-expression-profiling/)