Rna pico chip kit

After FACS sorting, RNA was extracted separately from GFP-positive and GFP-negative cells using an RNeasy Mini Kit (QIAGEN) and eluted in 20 μl of water with the eluate passed twice through the column to increase yield. The quantity of RNA was determined using a Nanodrop 2000c spectrophotometer (Thermo Fisher Scientific). Three biological replicates for the GFP-positive and GFP-negative samples (at ~50 ng/sample) were submitted to the Centre for Applied Genomics (Toronto, Canada). Here, the quality of the samples was assessed using the Agilent Bioanalyzer 2100 with the RNA Pico Chip kit (Agilent Technologies). RNA integrity number values between 6.5 and 7 were achieved. Expression profiling was performed according to the manufacturer’s instructions with Affymetrix GeneChip Mouse Gene 2.0 ST Array. Primary data analysis was carried out with the Affymetrix Expression Console, version 1.4.1.46, software, including the Robust Multiarray Average module for normalization. Gene expression data were log transformed. A change was considered significant when the FDR-corrected P value/q value thresholds met the criterion q < 0.01 at fold changes greater than 2 (expression increments or declines larger than 2). Raw sequence data were deposited in the NCBI’s Gene Expression Omnibus (GEO GSE201227).

All larvae and juveniles were preserved in RNAlater and stored at −20 °C until library preparation. Larvae and juveniles were both preserved at the ambient and heat treatments once the heat treatment reached ~50% mortality. Ten pooled larvae and two juveniles were extracted per cross/treatment due to low RNA concentrations per larva and juvenile. Extractions were performed using an RNAqueous kit (Ambion) and homogenized with an additional bead-beating step in 100 µl Lysis solution with ~6 glass beads (MPBio lysing matrix C) on the FastPrep (5.5 m/s for 30 sec × 2 times). Total RNA quantity was normalized to 375 ng for larval samples and 30 ng for juvenile samples using an RNA Pico Chip kit on the Agilent 2100 Bioanalyzer. Due to the high mortality of juveniles infected with C1 in the heat treatment, there was not adequate RNA to sequence these individuals. Samples were then selected and normalized based on RNA concentration and RIN (15 ng/µl for larvae, mean RIN = 6.6 and 2.5 ng/µl for juveniles, mean RIN = 6.9) and sent to Genomic Sequencing and Analysis Facility at the University of Texas at Austin for custom library preparation and SR50 tag-based sequencing on the Illumina Hi-Seq 2500.