title: GSM1179583: Axenic Amastigotes from CL causing Ldo isolate; Leishmania donovani; RNA-Seq
library_construction_protocol: Promastigotes total RNA were extracted from late log phase promastigotes culture; 2. Axenic amastigotes total RNA were extracted from axenic amastigotes culture twenty two hours after inoculating promastigotes in axenic amastigotes culture; 3. Infected B10R macrophages. B10R macrophages were cultured in DMEM medium supplemented with 10% heat-inactivated fetal bovine serum, 40 mM Hepes (pH 7.4),  50 U/ml Penicillin and 50 µg/ ml Streptomycin in a 37°C, 5% CO2 incubator. CL and VL axenic amastigotes from overnight axenic amastigotes culture were used to infect B10R cells at an amastigote / macrophage ratio of 10/1 in suspension  in DMEM medium overnight. The Leishmania infection levels were examined before extracting total RNA from these infected B10R cells. The percentage of infected macrophages is 72% for CL and 95% for VL; the average amastigotes number in an infected B10R cell is 3.6 for CL and 3.3 for VL. The infected B10R macrophage cells were harvested at 1200 RPM for 5 minutes to remove the uninfected Leishmania cells and the cell pellets were directly suspended in Trizol reagent for total RNA extraction. SL RNA-seq libraries were prepared using a protocol adapted from Armour et al. 2009. First strand cDNA was synthesized from 1 μg of DNase-treated total RNA using Superscript III (Invitrogen) and primer 1 (5'-TCCGATCTCTNNNNNNN-3'). The reaction was performed by incubating for 90 min at 40°C followed by 15 min at 70°C.The samples were then treated with 2U RNase H (Invitro- gen; 37°C for 20 min) to remove the RNA component of the RNA/DNA heteroduplex, and the resulting first strand DNA was purified on a QIAquick PCR purification column (QIAGEN). Second strand synthesis reaction was performed using the first strand DNA as template in a reaction mix containing a primer (5'-TCAGTTTCTGTA-3') that matches the Leishmania SL sequence and 3'-5' exo- Klenow Fragment (New England Biolabs, Inc.) according to the manufacturer’s protocol (incubation at 37°C for 30 min). The resulting double-stranded DNA was purified using the QIAquick PCR purification kit. Enrichment of DNA fragments with adapters was performed by PCR amplification of the purified double stranded DNA samples using the Expand Plus High Fidelity PCR System (Roche) and primers (5'-CAAGCAGAAGACGGCATACGAGCTCTTCCGATCTCT-3' and 5'-AATGATACGGCGACCACCGACACTCTTTCCCTACATCAGTTTCTGTACTTTA-3').A 2-min denaturation step at 94°C followed by two cycles (94°C for 10 s, 40°C for 2 min, 72°C for 1 min) and an additional 13–16 cycles (94°C for 10 s, 60°C for 30 s, and 72°C for 1 min) with a final 5-min, 72°C elongation step were performed. The resulting enriched fragments were purified on a QIAquick PCR purification column and represented the SL RNA library.