GSM5173330: WT 1; Plasmodium berghei; RNA-Seq
The infected blood was collected from mice and passed though Plasmodipure (Euro Proxima, Netherlands, cat# 8011) to remove WBCs. The purified RBCs were lysed in 0.2% saponin and the parasite pellet was washed thrice with PBS to obtain pellet devoid of any RBC contamination. The parasite RNA was isolated using Trizol and confirm RINs value above 8.0. Alternatively, the quality of the ribosomal 28 S and 18 S on an agarose gel was used as a measure of integrity and the absorbance at 260/280 nm was used to determine quantity. The sequencing library is prepared by random fragmentation of the cDNA sample preparation kit (TruSeq RNA Sample Preparation v2 Guide, Part #15026495 Rev. F.) according to the manufacturer's instructions. Briefly the total RNA (5µg) were ligated 5' and 3' adapter using T4 RNA ligase 2 truncated (NEB)and a 5' and 3' adapter using  T4 RNA ligase 2. The ligation products were reverse transcribed using Superscript II Reverse Transcriptase (Life Technologies) and amplified with  PCR (12 Cycle). The PCR products were purified with electrophoresis of 6% Novex TBE PAGE Gel. The purified cDNA library for each sample was analyzed on Agilent 2100 Bioanalyzer using Agilent DNA high sensitivity kit.