title: GSM8822276: BCG 72h -2; Mus musculus; RNA-Seq
library_construction_protocol: Total RNA was isolated from the infected BMDMs using EZ-10 DNAaway RNA Mini-Preps Kit (Bio Basic, Canada). TapeStation (Agilent Technologies 4200) was used to analyze RNA integrity of the preparation, and RNA quantification was done using Nanodrop (Thermo Scientific, USA). After the QC procedures, 4 ug of total RNA with >9 RIN (RNA Integrity Number) was used for RNA-Seq library preparation. PolyA RNA was enriched using oligo (dT) beads. PolyA RNA was then fragmented by heating to 94°C for 6 min. The first-strand cDNA was synthesized by SuperScript III reverse transcription kit (Invitrogen) using random hexamers, in the presence of RNasin Plus RNase Inhibitor, Actinomycin D, and DTT. Second-strand cDNA was synthesized with dUTP for strand-specific RNA sequencing, with DNA polymerase I (Enzymatics) in the presence of RNase H. Library preparation continued with end repairing, dA tailing for adapter ligation, and library purification with SparQ PureMag beads. Purified double-stranded cDNA libraries were lastly PCR-enriched and final purification. Quality control of the library consists of three steps: Quibit 2.0 (in-house) and q-PCR (at Novogene Inc.) for library quantification, and using Agilent 2100 (in-house & at Novogene Inc.) for library integrity and purity check. At Novogene Inc (Sacramento, CA), qualified libraries were fed into Illumina sequencers (NovaSeq 6000) for polyA RNA sequencing after pooling according to their effective concentration and expected data volume. Some libraries got sequenced twice due to insufficient data output in the first trial.