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Sample GSM3038538

Query DataSets for GSM3038538

Status

Public on Mar 12, 2018

Title

CandidaOnly_t0_repa

Sample type

SRA

Source name

C. albicans

Organism

Candida albicans

Characteristics

strain: SC5314-GFP-mCherry
genotype: SC5314-Neut5L-GFP-mCherry C. albicans

Treatment protocol

Prior to collection, macrophages were grown alone in RPMI, C. albicans was grown alone in RPMI or C. albicans and macrophages were miexed together and collected over time.

Growth protocol

Prior to collection, samples were grown at 37 degrees in 5% CO2 for either 0, 1, 2 or 4 hours in RPMI 1640 media

Extracted molecule

total RNA

Extraction protocol

Subpopulation samples were harvested, collected via FACs, flash frozen on dry ice, and RNA was extracted after bead mill lysis in tubes containg RLT and BME and the Qiagen Rneasy mini kit. Single, C. albicans infected libraries were sorted into wells of a 96 well plate containining RLT and BME, and frozen at -80 utnil cDNA synthesis. Illumina TruSeq RNA Sample Prep Kit (Cat#FC-122-1001) was used with 1 ug of total RNA for the construction of sequencing libraries.
RNA libraries were prepared for sequencing using standard Illumina protocols
Smart-seq2 was used to create cDNA, Nextera XT kits were used to make libraries;subpopulation and single infect cells were sequenced on Illumina's Nextseq. Candida only samples were sequenced on Illumina's Miseq

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina MiSeq

Description

C. albicans not exposed to macrophages and not sorted
CA_subpops_gene_exp_matrix

Data processing

QC and file trimming and sample demultiplexing was done with Picard version 1.107 and Trimmomatic.
BWA alignment version 0.7.10-r789.) to mouse transcriptome GRCm38/mm10 , the C. albcains transcriptome SC5314 version A21-s02-m09-r1 or a combined transcriptome containing both of these referenes ; Multi-reads (reads that aligned to both host and pathogen transcripts) were discarded.
Reads Per Kilobase of exon per Megabase of library size (RPKM) were calculated using a protocol from Chepelev et al., Nucleic Acids Research, 2009. In short, exons from all isoforms of a gene were merged to create one meta-transcript. The number of reads falling in the exons of this meta-transcript were counted and normalized by the size of the meta-transcript and by the size of the library.
Each host or pathogen sample file were aligned to its corresponding reference using Bowtie2 and RSEM (v.1.2.21).
For subpopulation samples, transcript abundance was estimated using transcripts per million (TPM). For subpopulation samples, TPM was calculated using edgeR in the Trinity package version 2.1. Genes were considered differentially expressed only if they had a 4-fold change difference (> 4 FC) in TPM values and a false discovery rate below or equal to 0.001 (FDR < 0.001), unless specified otherwise. For single macrophages infected with C. albicans, samples were aligned to the combined transcriptome as described above and RSEM was used to calculate TPM.
Genome_build: mouse transcriptome GRCm38/mm10; C. albcains transcriptome SC5314 version A21-s02-m09-r1 ; or a combined transcriptome made from mergeing these files
Supplementary_files_format_and_content: TPM matrix

Submission date

Mar 12, 2018

Last update date

Mar 14, 2018

Contact name

Toni Marie Delorey

E-mail(s)

delorey@broadinstitute.org

Phone

978-8528-8401

Organization name

Broad Institute