Entamoeba histolytica inter-species experiment transcriptome replicate 2
All strains were cultured in axenic media. E. histolytica strain HM1: IMSS was cultured in TYI-S-33 medium at 37C; E. moshkovskii strain Laredo was cultured in LYI-S-2 medium at room temperature; E. dispar strain SAW 760 was cultured in LYI medium at 37C and E. invadens strain IP-1 was cultured in TYI-S-33 medium at 25 C. Total RNA was extracted from approximately 1106 of trophozoites (with each culture performed in triplicates) of using Trizol (Invitrogen). The polyA fraction was purified from 10 to 100 g of total RNA using Dynabeads according to the manufacturers instructions (Thermofisher ref. 28152103011150). All libraries were purified on a 5% TBE gel (Biorad, # 456-5013) and were quantified by Bioanalyzer DNA High Sensitivity Chips (Agilent, # 5065-4626). Sequencing was performed on HiSeq-2000 (Illumina) in a multiplexed 51+7 nucleotide single-end read using a TruSeq SR Cluster kit v3 cBot HS (Illumina, # GD- 401-3002) and a TruSeq SBS kit v3 HS 50 cycles (Illumina, # FC-401-3002).  FASTQ sequence files were generated using CASAVA 1.8.2 (Illumina) and adapter sequences with trimmed using AlienTrimmer [23912058] (version 0.4.0). Reads shorter than 20 nucleotides were discarded.