title: GSM1835203: 4_1; Chlamydomonas reinhardtii; RNA-Seq
library_construction_protocol: Cell pellets were resuspended in 0.25 mL RNase-free water and then mixed with 0.25 mL warmed (70 C) lysis buffer (50 mM Tris-HCl pH 8.0, 200 mM NaCl, 20 mM EDTA, 2% SDS, 1 mg/mL Proteinase K). 10 mL TRIzol was then added and samples were mixed, flash frozen in liquid nitrogen, and stored at -80 C. Samples were later thawed and transfered to 15 mL MaXtract HD tubes followed by extraction with 2 mL chloroform. The aqueous phase was mixed with 1.5 volcumes 100% ethanol and applied to a MicroRNeasy column under vacuum. Washing, DNase treatment, and elution were performed according to manufacturer's instructions (RNase-free DNase Set). Plate-based RNASeq with polyA selection sample prep was performed on the PerkinElmer Sciclone NGS robotic liquid handling system, where purified mRNA is converted into cDNA library templates of known strand origin for sequencing on the Illumina Sequencer platform. The DynaBead kit (Invitrogen) was used to select and purify poly-A containing mRNA from 5 µg of total RNA per sample. The mRNA was chemically fragmented with 10X fragmentation solution (Ambion) at 70°C for 3 minutes to generate fragments ranging in size between 250 to 300bp. The fragmented RNA was purified using AMpure SPRI beads (Agencourt) using a ratio of 160:100 beads volume: RNA sample volume. First-strand cDNA was synthesized using SuperScript II Reverse Transcriptase (Invitrogen) and random hexamer primers (MBI Fermentas) with Actinomycin D in the master-mix to inhibit DNA dependent DNA synthesis. First-strand synthesis thermocycler conditions were 42°C for 50 minutes and an inactivation step at 70°C for 10 minutes. This was followed by another purification using AMpure SPRI beads at a ratio of 140:100 beads volume:cDNA volume. Second-strand cDNA synthesis was performed using DNA Polymerase I (Invitrogen), RNase H (Invitrogen), and a nucleotide mix containing dUTP (Roche). The second-strand synthesis was done at 16°C for 60 minutes. The double stranded cDNA fragments were purified using AMpure SPRI beads at a ratio of 75:100 beads volume: cDNA volume followed by a second purification using a bead:cDNA ratio of 140:100. cDNA fragments were end repaired, phosphorylated and A-tailed using a Kapa Biosystems kit followed by ligation to Illumina barcoded sequencing adapters. AmpErase UNG (Uracil N-glycosylase, Applied Biosystems) was added to the double stranded cDNA library fragments and incubated at 37°C for 15 minutes to cleave and degrade the strand containing dUTP. The single stranded cDNA was then enriched using 10 cycles of PCR with Illumina TruSeq primers and purified using AMpure SPRI beads at a ratio of 90:100 beads volume: cDNA volume to create the final cDNA library. Libraries were quantified using KAPA Biosystem’s next-generation sequencing library qPCR kit using a Roche LightCycler 480 real-time PCR instrument. The libraries were then prepared for sequencing on the Illumina HiSeq sequencing platform utilizing a TruSeq paired-end cluster kit, v3, and Illumina’s cBot instrument to generate clustered flowcells for sequencing. Sequencing of the flowcells was performed on the Illumina HiSeq2000 sequencer using a TruSeq SBS sequencing kit with 200 cycles, v3, following a 2x100 indexed run recipe.