GSM1642737: E. invadens trophozoite ~27nt small RNA; Entamoeba invadens; ncRNA-Seq
At each time point, parasites were harvested by chilling on ice, spun down, and washed 1x in cold PBS.  All samples were lysed using a French press at 400psi. Following lysis, RNA was isolated using Trizol (Invitrogen) following the manufacturer's protocol. We used the NEBNext multiplex small RNA library prep set for Illumina (NEB#E7300S) and followed the manufacturer’s protocol with the following modifications: total RNA (25-50 µg) was first fractioned for the size 15-30nt RNA using 12% polyacrylamide urea gel. The size-selected RNA was then treated with tobacco acid pyrophosphatase [TAP] to convert 5’ poly-P to 5’ single-P (Epicentre). The RNA sample was then fed into the pipeline as described in the manufacturer's protocol. Once the cDNA was made, the barcode primers were incorporated into each sample using PCR with kit’s provided barcoded primer; typically we used 8-12 PCR cycles to get a clear final PCR product band. The final PCR product was quantified using Nanodrop, and samples were pooled and shipped to the collaborator for Illumina sequencing. The platform MiSeq (Illumina) was used, the number Sequncing of small  RNAs during development