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Sample GSM1835248

Query DataSets for GSM1835248

Status

Public on Jul 30, 2015

Title

17_2

Sample type

SRA

Source name

Synchronized diurnal cullture

Organism

Chlamydomonas reinhardtii

Characteristics

strain: 6145c (CC-2895)
replicate time course: Replicate 2
protocol: dark
time (zeitgeber): ZT17

Treatment protocol

30 mL of bioreactor culture was sampled at indicated times and collected by centrifugation.

Growth protocol

Cells were innoculated from a plate into a 400 mL PSI FMT-150 photobioreactor cuvette containing HSM medium and grown in 12:12 light-dark conditions (125 uE red light, 125 uE blue light) at 28 degrees C. The culture was grown to a density corresponding to OD680=0.3 (~2-3e6 daughter cells/mL) and maintained at this density for 3-4 days before sampling.

Extracted molecule

total RNA

Extraction 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.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2000

Data processing

Quality control of RNA-Seq samples was performed on the raw paired-end reads using Trimmomatic to remove contaminating Illumina adapter sequences and low quality sequences (average Q20 over 4 base slide window<20).
Reads were aligned to the Chlamydomonas reinhardtii genome version 5 assembly with STAR version 20201 using standard presets except for intron size that was set between 20 and 3,000 bp (--alignIntronMin 20 & --alignIntronMax 3000).
Uniquely mapping reads were assigned to 17,737 version 5.3.1 primary transcripts using HT-Seq version 0.5.4p3 (htseq-count --mode intersection-nonempty --type exon --stranded=no).
One read was added to each sample/gene locus and samples were normalized by library size (# uniquely-mapping reads). Differential expression analysis was performed using DESeq2 version 1.4.5 comparing samples to mean expression of all samples. HTSFilter version 1.4.0 was used to establish a low-expression filter of 1.061 reads/million (RPM) and genes with less that 1.061 RPM average expression were excluded from multiple testing correction (FDR). Genes with an absolute fold-change ≥ 2 compared to the mean expression and with a FDR < 0.05 in at least one sample were considered differentially expressed. Expression estimates were then length normalized by primary transcript length into RPKMs.
Expression estimates were transformed to into standard deviation/mean (z-score). Then K-means Support clustering was performed by MeV version 4.9 on differentially expressed genes to obtain 6 groups of genes with similar expression patterns. These gene sets were then clustered again to obtain a total of 18 groups of differentially-expressed genes.
Genome_build: Chlamydomonas reinhardtii version 5.
Supplementary_files_format_and_content: ChlamydomonasSynchronousDiurnalExpressionHTSeqCounts.txt - Tab-delimited text file, HTSeq read counts
Supplementary_files_format_and_content: ChlamydomonasSynchronousDiurnalExpressionRPKM.txt - Tab-delimited text file, RPKM expression estimates

Submission date

Jul 29, 2015

Last update date

May 15, 2019

Contact name

James Umen

E-mail(s)

jumen@danforthcenter.org

Phone

314-587-1689

Organization name

Donald Danforth Plant Science Center