Eimeria tenella noncoding RNA sequencing - sporozoites at 28 days elapsed since beginning of sporulation
Description
Noncoding RNA sequencing for 28 day old Eimeria sporozoites (i.e., sporulated oocysts). All oocysts were stored at room temperature (21 C) from the beginning of sporulation. NS.1389.001.NEBNext_Index_43.2HB_R1.fastq - 28 day old sporulated E. tenella oocysts, warmed to 41 C for 90 minutes prior to RNA recovery - read 1 NS.1389.001.NEBNext_Index_43.2HB_R2.fastq - 28 day old sporulated E. tenella oocysts, warmed to 41 C for 90 minutes prior to RNA recovery - read 2 NS.1389.001.NEBNext_Index_44.2RD_R1.fastq - 28 day old sporulated E. tenella oocysts, held at steady state (21 C) prior to RNA recovery - read 1 NS.1389.001.NEBNext_Index_44.2RD_R2.fastq - 28 day old sporulated E. tenella oocysts, held at steady state (21 C) prior to RNA recovery - read 2
Files
Steps to reproduce
RNA was recovered from sporulated oocysts 28 days following collection from two parallel samples of 30 million oocysts: one that been stimulated by warming to 41C for 90 minutes prior to RNA recovery, and a second that remained at steady state (21C). Briefly, oocysts were washed in ddH2O and were subsequently resuspended in 300 μL buffer RLT (Qiagen, Hilden, Germany) with dithiothreitol (DTT; 40 mM) and were disrupted mechanically at three × 30 seconds with 5 second pauses at 6000 rpm in a Precellys® bead mill homogenizer. Then 150 μL Buffer RLT (+40 mM DTT) was added prior to further processing using Qiagen QIAshredder columns, as per manufacturer’s instructions. One volume of 70% EtOH was added to flowthrough and material was transferred to the RNeasy® spin column included in Qiagen RNeasy® Mini kit. Subsequent steps were as per manufacturer’s instructions. Approximately 1200 ng of RNA from each sample was submitted to Genome Quebec (Canada) for library construction and NGS. Stranded libraries were prepared using NEBNext® Multiplex Small RNA Prep Kit for Illumina with no size selection. NEBNext adaptors and indexing primers were used. One quarter of a sequencing lane was dedicated to each sample. Paired end, 100 base reads were generated on an Illumina NovaSeq 6000 Sequencing System.