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| Status |
Public on May 03, 2022 |
| Title |
Candida albicans, CRISPRa non-targeting control, rep3 |
| Sample type |
SRA |
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| Source name |
Total
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| Organism |
Candida albicans |
| Characteristics |
tissue: Total
cell line: SC5314
genotype: CRISPRa Control (WT with Non-Targeting CRISPRa Construct)
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| Growth protocol |
All strains were grown in YPD at 30°C.
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| Extracted molecule |
total RNA |
| Extraction protocol |
RNA extraction and sample QC was conducted at GENEWIZ, LLC./Azenta US, Inc (South Plainfield, NJ, USA) as follows: Total RNA was extracted using Qiagen RNeasy Plus Universal kit following manufacturer’s instructions (Qiagen, Hilden, Germany). RNA samples were quantified using Qubit 2.0 Fluorometer (ThermoFisher Scientific, Waltham, MA, USA) and RNA integrity was checked with 4200 TapeStation (Agilent Technologies, Palo Alto, CA, USA).
Library preparations and sequencing reactions were conducted at GENEWIZ, LLC./Azenta US, Inc (South Plainfield, NJ, USA) as follows: ERCC RNA Spike-In Mix kit (cat. 4456740) from ThermoFisher Scientific, was added to normalized total RNA prior to library preparation following manufacturer’s protocol. The RNA sequencing library was prepared using the NEBNext Ultra II RNA Library Prep Kit for Illumina using manufacturer’s instructions (New England Biolabs, Ipswich, MA, USA). Briefly, mRNAs were initially enriched with Oligod(T) beads. Enriched mRNAs were fragmented for 15 minutes at 94 °C. First strand and second strand cDNA were subsequently synthesized. cDNA fragments were end repaired and adenylated at 3’ends, and universal adapters were ligated to cDNA fragments, followed by index addition and library enrichment by PCR with limited cycles. The sequencing library was validated on the Agilent TapeStation (Agilent Technologies, Palo Alto, CA, USA), and quantified by using Qubit 2.0 Fluorometer (ThermoFisher Scientific, Waltham, MA, USA) as well as by quantitative PCR (KAPA Biosystems, Wilmington, MA, USA). The sequencing libraries were multiplexed and clustered onto a flowcell. After clustering, the flowcell was loaded onto the Illumina HiSeq 4000 or equivalent instrument according to manufacturer’s instructions. The samples were sequenced using a 2x150bp Paired End (PE) configuration. Image analysis and base calling were conducted by the Illumina Control Software. Raw sequence data (.bcl files) generated from the Illumina instrument was converted into fastq files and de-multiplexed using Illumina bcl2fastq 2.20 software. One mis-match was allowed for index sequence identification.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 4000 |
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| Data processing |
Sequence reads were trimmed to remove possible adapter sequences and nucleotides with poor quality using Trimmomatic v.0.36.
The trimmed reads were mapped to the candida_albicans reference genome available on ENSEMBL using the STAR aligner v.2.5.2b. The STAR aligner is a splice aligner that detects splice junctions and incorporates them to help align the entire read sequences. BAM files were generated as a result of this step.
Unique gene hit counts were calculated by using featureCounts from the Subread package v.1.5.2. The hit counts were summarized and reported using the gene_id feature in the annotation file. Only unique reads that fell within exon regions were counted.
After extraction of gene hit counts, the gene hit counts table was used for downstream differential expression analysis. Using DESeq2, a comparison of gene expression between the control and experimental groups of samples was performed. The Wald test was used to generate p-values and log2 fold changes. Genes with an adjusted p-value < 0.05 and absolute log2 fold change > 1 were called as differentially expressed genes for each comparison.
Assembly: Candida albicans SC5314 (assembly ASM18296v3).
Supplementary files format and content: Tab-delimited text files including transcript hit counts .
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| Submission date |
Apr 29, 2022 |
| Last update date |
May 03, 2022 |
| Contact name |
Rebecca S Shapiro |
| E-mail(s) |
shapiror@uoguelph.ca
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| Organization name |
University of Guelph
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| Department |
Molecular and Cellular Biology
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| Lab |
The Shapiro Lab
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| Street address |
50 Stone Road East
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| City |
Guelph |
| State/province |
ON |
| ZIP/Postal code |
N1G 2W1 |
| Country |
Canada |
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| Platform ID |
GPL24129 |
| Series (1) |
| GSE201904 |
Design principles and applications of a CRISPR activation system for facile genetic overexpression in Candida albicans |
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| Relations |
| BioSample |
SAMN27992872 |
| SRA |
SRX15053916 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6077776_Resub-D3.counts.txt.gz |
106.6 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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