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| Status |
Public on Sep 26, 2017 |
| Title |
WT2 Run 2 |
| Sample type |
SRA |
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| Source name |
cultured cells
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| Organism |
Cryptococcus neoformans |
| Characteristics |
strain: KN99
genotype: wildtype
condition description: Capsule-inducing
condition: 37C.CO2.90m
replicate: 2
induction date: 03.31.17
library date: 04.05.17
sample id: 584
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| Treatment protocol |
Cells were transfered to grow for 90 minutes in either capsule-inducing (DMEM, 37 °C, 5% CO2) conditions prior to isolation of polyA RNA.
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| Growth protocol |
All strains were constructed in the C. neoformans KN99α background. Cells were cultured overnight in YPD to a density of 1-2 OD600.
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
RNA is isolated from liquid culture following the protocol for TRIzol RNA Isolation Reagent (Life Technologies 15596026). Poly-A RNA is separated using an mRNA Catcher Plus Plate (Invitrogen K1570-02). Once the library is made it is sequenced on a HiSeq 2500 by Illumina.
The libraries are made by chemically shattering the RNA in TURBO DNase I (Invitrogen NC9075048) buffer at 75 degrees for 10 min. To make first-strand cDNA, shattered genomic DNA is incubating with Random Primers (Invitrogen 48190-011) and dNTP mix (NEB N0447S) at 65 degrees for 5 min. The first strand is then synthesized by using SSIII kit (Invitrogen 18080-44) 5X First Strand Buffer, DTT, and SSIII enzyme; and adding RNase OUT (Invitrogen 10777-019). It is incubated as follows: 25 degrees for 5 min, 50 degrees for 50 min, 70 degrees for 15 min, and holding at 4 degrees.The second strand is made by adding 5X SS buffer (Invitrogen 10812-014), dNTP mix (NEB N0447S), E. coli DNA ligase (NEB M02056), DNA Polymerase I (Invitrogen 18010-025), RNase H (Invitrogen 18021-014) and incubating at 16 degrees for 2 hours. Then end repair is carried out using the NEB Quick Blunting Kit (E1201S), adding an A base using Klenow Fragment (3’ to 5’ exo, NEB M0212S) and 1mM dATP (Invitrogen 18252-015), and finally adding the Illumina adapters and indices.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
Grown in 37°C + 5% CO2 + high glucose DMEM for 90 min
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| Data processing |
Sequenced reads were aligned to the C. neoformans H99 reference sequence (v3) using TopHat version 2.0.14 and Bowtie version 0.12.9
Reads that aligned uniquely to the reference sequence were considered for gene expression quantification with HTSeq version 0.6.1. Gene boundaries were defined using the current C. neoformans genome annotation provided by the Broad institute.
Differential expression analysis comparing matched mutant and wild type expression profile replicate sets was performed with DESeq version 1.24.0 built under R 3.3.0 using an adjusted p-value threshold of 0.01. Expression was normalized using the DESeq standard estimateSizeFactors procedure.
Genome_build: C. neoformans H99 reference sequence 3.0 (Cryptococcus neoformans var. grubii H99 Sequencing Project)
Supplementary_files_format_and_content: Gene expression was quantified processing Tophat produced bam alignments with HTSeq 0.6.1 with the current H99 annotation (v3) provided by the Broad institute's Fungal Genome initiative. Expression was normalized with DESeq version 1.24.0 built under R 3.3.0 using the estimateSizeFactors procedure.
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| Submission date |
Sep 25, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Ezekiel Maier |
| E-mail(s) |
emaier@wustl.edu
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| Organization name |
Washington University in Saint Louis
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| Department |
Computer Science
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| Lab |
Michael Brent
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| Street address |
4515 McKinley Ave
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| City |
Saint Louis |
| State/province |
MO |
| ZIP/Postal code |
63110 |
| Country |
USA |
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| Platform ID |
GPL19081 |
| Series (1) |
| GSE104198 |
Unintended side effects of transformation are very rare |
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| Relations |
| BioSample |
SAMN06842807 |
| SRA |
SRX3049053 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2791804_00000-37C.CO2.90m-2-03.31.17-04.05.17-584.counts.txt.gz |
28.5 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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