|
| Status |
Public on Mar 12, 2018 |
| Title |
SIC_InfectedMO_liveCA_t240_S194 |
| Sample type |
SRA |
| |
|
| Source name |
macrophage and C. albicans
|
| Organisms |
Candida albicans; Mus musculus |
| Characteristics |
strain: C57BL/6 and SC5314-GFP-mCherry
single infected macrophage plate number: 4
genotype: C57BL/6 primary, bone derived macrophages and 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 |
NextSeq 550 |
| |
|
| Description |
Sorted, single macrophages infected with live C. albicans
CA_singlecells_gene_exp_matrix
MO_singlecells_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
|
| Street address |
415 main street
|
| City |
cambridge |
| State/province |
ma |
| ZIP/Postal code |
02130 |
| Country |
USA |
| |
|
| Platform ID |
GPL24726 |
| Series (1) |
| GSE111731 |
Revealing parallel host-pathogen transcriptional dynamics using sorted subpopulations and single, Candida albicans infected macrophages |
|
| Relations |
| BioSample |
SAMN08688804 |
| SRA |
SRX3783479 |
