title: GSM6514321: PBS2hr rep 2; Mus musculus; RNA-Seq
library_construction_protocol: BMDMs (1 x10^6 cells/well) in each well of a 6-well culture plate were infected with MSC or MSM at an MOI of 1:5 or uninfected (control cells) and incubated for 2 h at 37°C in a CO2 incubator. After the incubation, the cells were washed with 1xPBS to remove the extracellular bacteria. Then the cells were lifted using cell scraper and collected via centrifugation at 200xg for 10 minutes. 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). 4 ug of total RNA with >9 RIN (RNA Integrity Number) was used for RNA-Seq library preparation. The quality assessment of RNA samples was carried out by Nanodrop, agarose gel electrophoresis, and Tape station. After the QC procedures, mRNA was enriched using oligo (dT) beads. First, the mRNA was fragmented randomly by adding a fragmentation buffer. Then the cDNA is synthesized by using an mRNA template and random hexamers primer, after which a custom second-strand synthesis buffer (Illumina), dNTPs, RNase H, and DNA polymerase I are added to initiate the second-strand synthesis. Second, after a series of steps, including terminal repair, A ligation, and sequencing adaptor ligation, the double-stranded cDNA library is completed through size selection and PCR enrichment. Quality control of the library consists of three steps: Quibit 2.0, Agilent 2100, and Q-PCR. Finally, qualified libraries are fed into Illumina sequencers (NovaSeq 6000) after pooling according to their effective concentration and expected data volume. RNA-sequencing was done at Novogene Corporation Inc. (Sacramento, CA, USA). Strand-Specific (dUTP-based) paired end libraries