title: GSM7431870: Eh control trophozoites, 231221; Entamoeba histolytica; RNA-Seq
library_construction_protocol: Trophozoites were incubated with different forms of Bacillus subtilis (planktonic or biofilm) or serum-free Diamond's TYI S-33 medium without bacteria as a control. The trophozoites were incubated for 1 hour at 37°C in a 24-well plate that was covered with serum-free Diamond's TYI S-33 agar. After the incubation period, the trophozoites were harvested by gently tapping the glass tube, followed by centrifugation at 1900 rpm for 3 minutes. RNA extraction from E. histolytica was performed using the Monarch Total RNA Miniprep Kit ( NEW ENGLAND BioLabs, Ornat, Nes Ziona, Israel). The extraction protocol was followed according to the manufacturer's instructions, which emphasized the importance of avoiding mechanical disruption during the cell lysis step to ensure the extraction of E. histolytica RNA while minimizing extraction of B. subtilis biofilm RNA using the detergent-based lysis buffer. Simultaneously, a total of 9 RNAseq libraries were constructed following the manufacturer's protocol (NEBNext Ultra II Directional RNA Library Prep Kit for Illumina, cat no. E7760). Each library was prepared using 400 ng of total RNA as the starting material. To enrich for mRNA, a magnetic pull-down method was employed using the Magnetic Isolation Module (NEB, cat no. E7490). Subsequently, the concentration of each library was measured using Qubit (Invitrogen), while the size distribution was determined using the TapeStation 4200 with the High Sensitivity D1000 kit (cat no. 5067-5584). To ensure equal representation, all libraries were combined into a single tube with equal molarity. The RNAseq data was generated on an Illumina NextSeq2000 platform using P2 chemistry with 100 cycles (Read1-100; Index1-8; Index2-8) (Illumina, cat no. 20046811).