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Sample GSM4988920

Query DataSets for GSM4988920

Status

Public on Dec 23, 2021

Title

dUTP strand-specific RNA-seq log-phase cells of Schizosaccharomyces japonicus grown in BMW

Sample type

SRA

Source name

log-phase cells

Organism

Schizosaccharomyces japonicus

Characteristics

media: BMW
growth temperature (celcius): 30
growth phase: log phase

Growth protocol

We conducted RNA-seq on the following 11 Ascomycota yeast species: Candida albicans, Candida glabrata, Kluyveromyces lactis, Kluyveromyces waltii, Saccharomyces bayanus, Naumovozyma castellii, Saccharomyces cerevisiae, Schizosaccharomyces japonicus, Schizosaccharomyces octosporus, Schizosaccharomyces pombe, Yarrowia lipolytica. Each of the 11 species was grown in BMW medium, chosen to minimize cross-species growth differences, as previously described (Thompson et al., 2013). N. castellii was grown at 25℃ while the rest of the species were grown at 30℃.

Extracted molecule

total RNA

Extraction protocol

RNeasy Midi or Mini Kits (Qiagen, Valencia, CA) were used to isolate total RNA from log-phase cells by mechanical lysis using the manufacturer instructions as previously described (Thompson et al., 2013).
dUTP strand-specific RNA-seq libraries were constructed as previously described (Yassour et al., 2010) with the following modifications. (1) The polyA+-selected RNA was fragmented in a 40 µl reaction containing 1x Fragmentation Buffer (Affymetrix) by heating at 80℃ for 4 minutes followed by cleanup via ethanol precipitation for all libraries (except Y. lipolytica, S. pombe, S. japonicus, and S. octosporus; for these species, the conditions described previously were used (Yassour et al., 2010)), followed by cleanup via 1.8x RNAClean XP beads (Beckman Coulter Genomics). (2) For C. glabrata, K. lactis, S. bayanus, S. pombe, S. japonicus, and S. octosporus libraries, the adapter ligation was performed overnight at 16℃. For the rest, this was done at 16 degrees for 2 hours as described previously (Yassour et al., 2010). (3) Normalization was carried out based on the cDNA input and pooling of selected Illumina barcoded-adaptor-ligated cDNA products followed by gel size selection occurred as follows: range of 275 to 575 bp for pooled C. albicans, K. polysporus, K. waltii, and N. castellii libraries, and 375 to 575 bp for C. glabrata, K. lactis, and S. bayanus libraries. For the other libraries, no pooling was performed before gel size-selection – range of 310 to 510 bp for Y. lipolytica and 350 to 550 bp for S. pombe, S. japonicus, and S. octosporus. (4) The final PCR product was purified by 1.8x AMPure XP beads (Beckman Coulter Genomics) followed by a second gel size-selection for the range of 300 to 575 bp for C. albicans, K. polysporus, K. waltii, and S. castellii libraries, but no gel size-selection was performed for the other libraries. The pooled final library was sequenced on one to four lanes of HiSeq2000 (Illumina) with 68 base (Y. lipolytica had 76 base) paired-end reads and 8 base index reads.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2000

Data processing

For each of the 11 Ascomycota yeast species described above, the reads were merged and assembled using Trinity (Grabherr et al., 2011) (version ‘trinityrnaseq_r2012-05-18’) and mapped onto the assemblies to the respective genomes using GMAP (Wu and Watanabe, 2005). The Jaccard coefficient was used to join adjacent assemblies given enough connecting reads (a Jaccard cutoff of 0.35, the default in Trinity was used). Finally, upon mapping all of the reads to all of the merged assemblies, the Jaccard coefficient was used to clip assemblies which did not have enough support over a certain region. For each of the species, the reads were mapped to the genome sequence (Wapinski et al., 2007) using BLAT (Kent, 2002).
The RPKM used in the analysis described below were calculated for each transcript using RSEM (Li and Dewey, 2011), and only reads mapping to the sense mRNA strand were considered. Orthology between genes in different species was used as determined previously (Wapinski et al., 2007).
Genome_build: Various (from Wapinski et al, 2007, Nature)
Supplementary_files_format_and_content: tab delimited text containing all RPKM values for genes with S. cerevisiae orthologs

Submission date

Dec 24, 2020

Last update date

Dec 23, 2021

Contact name

Carl G de Boer

Organization name

The Broad Institute

Lab

Aviv Regev

Street address

415 Main St