GSM2411758: M2_Media.24h_Donor.20; Homo sapiens; RNA-Seq
After removing cell culture supernatants, uninfected and BCG-infected macrophages were harvested in trizol. Chloroform (0.2 parts) was added; total RNA in upper phase of partition was precipitated with Isopropanol and washed with 80% Ethanol.  cDNA libraries were prepared using TruSeq RNA Library Preparation Kit v2 (RS-122-2001) according to the “TruSeq RNA Sample Preparation v2 Guide”. Briefly, purification of poly-A containing mRNA molecules from 300 ng of total RNA was performed using poly-T oligo attached magnetic beads. After fragmentation into small pieces using divalent cations under elevated temperature, the cleaved RNA fragments were copied into first strand cDNA using reverse transcriptase and random primers. Second strand cDNA synthesis was achieved using DNA Polymerase I and RNase H. These cDNA fragments went through further end repair process, single “A” base addition, and adapters ligation. The products were then purified and enriched with PCR to create the final cDNA library. Libraries were sequenced on Illumina HiSeq2000 at 50 million reads per sample and 1X50 read length. These procedures were performed by the UCLA Clinical Microarray Core facility (http://www.pathology.ucla.edu/cmc).