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Sample GSM2791807 Query DataSets for GSM2791807
Status Public on Sep 26, 2017
Title G418_6 2 Run 2
Sample type SRA
 
Source name cultured cells
Organism Cryptococcus neoformans
Characteristics strain: KN99
genotype: insertion of kanMX at chr11:287563
condition description: Capsule-inducing
condition: 37C.CO2.90m
replicate: 2
induction date: 03.31.17
library date: 04.05.17
sample id: 587
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.
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.
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.
 
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina HiSeq 2500
 
Description Grown in 37°C + 5% CO2 + high glucose DMEM for 90 min
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.
 
Submission date Sep 25, 2017
Last update date May 15, 2019
Contact name Ezekiel Maier
E-mail(s) [email protected]
Organization name Washington University in Saint Louis
Department Computer Science
Lab Michael Brent
Street address 4515 McKinley Ave
City Saint Louis
State/province MO
ZIP/Postal code 63110
Country USA
 
Platform ID GPL19081
Series (1)
GSE104198 Unintended side effects of transformation are very rare
Relations
BioSample SAMN06705766
SRA SRX3049116

Supplementary file Size Download File type/resource
GSM2791807_00000_6_G418-37C.CO2.90m-2-03.31.17-04.05.17-587.counts.txt.gz 28.6 Kb (ftp)(http) TXT
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Raw data are available in SRA
Processed data provided as supplementary file

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