title: RNA-Seq of daphnia magna whole body after 24 h exposure to FC10diol
design: At test termination, each well was inspected and all mortalities (immobile with gentle prodding) were recorded and removed. Exposure medium in each well was replaced with 60 l of Trizol (Zymo Research, CA, USA). A 2.3 mm stainless steel bead was added to each well, then the plate was homogenized at 30 Hz for two minutes using an Omni-Bead Ruptor bead mill. Homogenates of individual Daphnia magna from the same treatment group were pooled together, on a per plate basis (Figure S.2D) to generate one pooled sample per treatment, per plate. RNA was extracted from pooled homogenates using Zymo Research Direct-zol-96 RNA kit (Zymo Research) protocol adapted to the Biomek i5 to yield 40 l of RNA. Previously extracted control RNA was added to empty wells on the final RNA plate. Concentrations of RNA for each well were measured on a NanoDrop spectrophotometer (Thermo Scientific, NanoDrop Eight) and diluted if necessary. RNA extracts (10 l) were used to prepare cDNA libraries with the Lexogen Poly(A) RNA Selection Kit V1.5 (catalog #157; Lexogen GmbH, Austria) and Lexogen CORALL mRNA-Seq Library Prep Kit with UDIs (catalog #158-163) both adapted to the Biomek i5. Resulting cDNA library samples were evaluated on a 4200 TapeStation (Agilent Technologies, CA, USA) using the D1000 ScreenTape (Agilent) analysis protocol to assess cDNA quality. Samples were quantified using the Quant-iT Picogreen dsDNA assay kit (catalog # P11495; Thermo Fischer Scientific), diluted if necessary, and equal portions of each sample pooled for use in RNA sequencing. RNA sequencing was performed at the Michigan State University Research Technology Support Facility Genomics Core. Libraries were quality control checked and quantified using a combination of Qubit dsDNA HS, Agilent 4200 TapeStation HS DNA1000, and Invitrogen Collibri Library Quantification qPCR assays. Each pool (corresponding to  96 samples from assays with two PFAS) was loaded onto one lane of a NovaSeq S4 flow cell. Sequencing was performed in a 2x150bp paired end format using a NovaSeq 6000 v1.5 300 cycle reagent kit (target read depth of 28,000 MB/sample). Base calling was done by Illumina Real Time Analysis v3.4.4, and the output was demultiplexed and converted to FASTQ format with Illumina Bcl2fastq v2.20.0.
library_name: DMFC10diolP1CS72_L002