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Sample GSM4988916 Query DataSets for GSM4988916
Status Public on Dec 23, 2021
Title dUTP strand-specific RNA-seq log-phase cells of Kluyveromyces waltii grown in BMW
Sample type SRA
 
Source name log-phase cells
Organism Lachancea waltii
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
City Cambridge
State/province MA
ZIP/Postal code 02139
Country USA
 
Platform ID GPL29533
Series (1)
GSE163866 The evolution, evolvability, and engineering of gene regulatory DNA [RNA-seq]
Relations
BioSample SAMN17154390
SRA SRX9734586

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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