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Sample GSM3675053

Query DataSets for GSM3675053

Status

Public on Nov 13, 2019

Title

BTX642 Leaves, 70 days old, Control, plot 2

Sample type

SRA

Source name

Leaves

Organism

Sorghum bicolor

Characteristics

genotype: BTX642
watering regime: Control
age (days): 70
plot: 2

Extracted molecule

polyA RNA

Extraction protocol

Frozen root and leaf tissue were ground in a cryogenic Freezer Mill (SPEX SamplePrep 6875D, Metuchen NJ USA) for 2-3 cycles of 2-3 minutes, with 1 minute cooling in between. Samples were then stored at -80C. RNA was extracted using the QIAGEN miRNeasy mini kit (Cat. #AM217004) with modifications: 1 mL QIAzol (Cat) and 100 uL 10% Sarkosyl solution were added to 100 mg of frozen tissue and placed in a vortex shaker adapter for 5 minutes. To these, 200 uL chloroform was added, and the samples were then vortexed and incubated for 3 minutes at room temperature before centrifugation at 12,000 xg for 15 min at 4C. The aqueous phase was then transferred to a fresh tube, and ethanol was added to a final concentration of 60%. The remaining procedure was performed according to the manufacturer’s recommendations. DNA contamination was removed from each sample using the TURBO DNA-free kit, (Cat. # AM1907, Invitrogen) according to the manufacturer’s recommendations.
Stranded cDNA libraries were generated using the Illumina Truseq Stranded RNA LT kit. mRNA was purified from 1 ug of total RNA using magnetic beads containing poly-T oligos. mRNA was fragmented and reversed transcribed using random hexamers and SSII (Invitrogen) followed by second strand synthesis. The fragmented cDNA was treated with end-pair, A-tailing, adapter ligation, and 8 cycles of PCR. qPCR was used to determine the concentration of the libraries.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2500

Description

leaf_normCounts.txt
0810162L02

Data processing

Using BBDuk, raw reads were evaluated for artifact sequence by kmer matching (kmer=25), allowing 1 mismatch and detected artifact was trimmed from the 3'end of the reads.
RNA spike-in reads, PhiX reads and reads containing any Ns were removed.
Quality trimming was performed using the phred trimming method set at Q6.
Following trimming, reads under the length threshold were removed (minimum length 25 bases or 1/3 of the original read length - whichever is longer).
Filtered reads from each library were aligned to the reference genome (phytozome v3.1, supplemented with RTx430 and BTx642 SNP information) using HISAT version 2.1.0. featureCounts was used to generate the raw gene counts file using gff3 annotations. Only primary hits assigned to the reverse strand were included in the raw gene counts (-s 2 -p --primary options).
Genome_build: Sorghum bicolor v. 3.1 (JGI), supplemented with resequence data available in SRR4028151 (BTx642) or SRR4028129 (RTx430)
Supplementary_files_format_and_content: normalized counts for root and leaf tissues are provided separately as text files. The reference genome fasta files (SNP-corrected) are provided as fasta files.

Submission date

Mar 18, 2019

Last update date

Nov 13, 2019

Contact name

Benjamin Jeremy Cole

Organization name

DOE-Joint Genome Institute

Street address

1, Cyclotron Road

City

Berkeley