Name: GSM8114847
Instrument: Illumina NovaSeq 6000
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: PAIRED
Construction protocol: The cells were pelleted by centrifugation (4,300 x g for 10 minutes) at 4°C and stored at -80°C. Cell pellets were resuspended in 2 mL of lysis solution (2% sodium dodecyl sulfate, 16mM EDTA, RNase-free water) and heated at 65°C for 5 minutes. Nucleic acids were extracted using 2 mL of acidic phenol:chloroform heated to 65°C, mixed by inversion, incubated at 65°C for 5 minutes, and centrifuged at room temperature for 7 minutes at 20,000 x g. The aqueous phase was removed, extracted twice more, before addition of 2 mL of chloroform, mixed, and centrifuged once more at room temperature for 7 minutes at 20,000 x g. The aqueous layer was removed and mixed with 1/10 volume of 3M sodium acetate and equal volume of isopropanol, and incubated at -20°C for >1 hour to precipitate nucleic acids that were collected by centrifugation at 4°C for 30 minutes at 16,000 x g. The pellet was washed twice with 1 mL of 75% EtOH (prepared with RNase-free water). Once residual ethanol was removed by evaporation, the pellet was resuspended in 85 μL of RNase-free water. Samples were treated with RNase-free DNase (10 μL 10x DNase buffer, 2 μL DNase, 3 μL RNasin (Promega)) and purified using RNeasy kit (Qiagen). RNA-seq library preparation and sequencing was performed by the Joint Genome Institute (JGI) using default parameters. rRNA in the samples was depleted using the QIAseq FastSelect kit (Qiagen, Germantown, MD). Libraries were constructed using the TruSeq stranded mRNA kit (Illumina) following standard JGI protocols.