Stranded RNA-seq of LdoS-infected macrophages after 24 hr (rep 1)
THP1 macrophages were grown in 8 plates in 37C at a density of 1x107                 cells per plate. Mid log-phase L. donovani promastigotes were added to THP1 culture                 for 4 hours at a ratio of 1:10 macrophage to parasite. Subsequently, plates were                 washed three times with room temperature PBS and the incubated for 48 hours in RPMI                 1640 growth medium. Macrophages, infected or not, were collected with a scraper and                 washed with ice-cold PBS for 4 minutes at 48 X g. The pellets were then resuspended                 in 1 mL PBS and transferred to Eppendorf tubes. Separation of amastigotes was                 facilitated initially by passing the pellet repeatedly through a 1 mL syringe with                 27G needle. Lysates were then centrifuge 15 minutes at 2369Xg at 4C. Pellet was then                 resuspemded in PBS and overlayed on1 ml Percoll (****). Cells were then centrifuged                 Logarithmically-growing cells were collected and resuspended at a concentration of                 5x108 cells/mL in TriZOL (Invitrogen). The aqueous phase was isolated by adding 0.2                 mL chloroform (Fisher Scientific) and centrifugation at 12,000 x g for 15 minutes at                 4C. The aqueous phase was isolated and RNA was precipitated by adding 1 volume of                 100% isopropanol (Fisher Scientific) and centrifugation at 12,000 x g for 10 minutes                 at 4C. The RNA pellet was washed with 75% ethanol (Pharmco) and was resuspended in                 nuclease-free water (Ambion). Purified RNA was treated with 20 units of DNase I                 (Ambion) and was precipitated using 1/10 volumes 3M sodium acetate (Sigma) and 3                 volumes 100% ethanol. RNA was pelleted by centrifugation at 15,000 g-1 and the                 resulting pellet was washed with cold 75% ethanol and resuspended in RNAse-free                 water (Ambion). TruSeq Stranded Total RNA Library Prep Kit from illumina and its                 standared protocol was used to prepare RNAseq libraries from total                 RNA.