GSM1838792: Macrophage Amastigote (intracellular) stage cells with no arginine treatment rep2; Leishmania donovani; RNA-Seq
Extraction of intracellular parasites from macrophage cells: THP1 macrophages were grown in 8 plates in 37°C 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 4˚C. 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 4°C. 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 4°C. 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.
GEO Accession
GSM1838792