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| Status |
Public on Dec 06, 2021 |
| Title |
1494_1_PMBC_unstim |
| Sample type |
SRA |
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| Source name |
PBMC
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| Organism |
Homo sapiens |
| Characteristics |
cell type: PBMC
stimulation: Unstim
bcg state: pre
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| Treatment protocol |
PBMCs were stimulated with a Mtb-derived epitope pool (MTB300), BCG bacteria, or left unstimulated for 24 hours before sorting.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was purified using an miRNeasy Micro Kit (QIAGEN) and quantified by quantitative PCR. Purified total RNA (1-5 ng) was amplified following the Smart-Seq2 protocol.
mRNA was captured using poly-dT oligonucleotides and directly reverse transcribed into full-length cDNA (amplified by PCR for 16 cycles). cDNA was purified using AMPure XP beads (Beckman Coulter). From this step, 1 ng of cDNA was used to prepare a standard Nextera XT sequencing library (Nextera XT DNA sample preparation kit and index kit, Illumina). Whole-transcriptome amplification and sequencing library preparations were performed in a 96-well format to reduce assay-to-assay variability. Barcoded Illumina sequencing libraries (Nextera, Illumina) were generated using the automated platform (Biomek FXp). Libraries were sequenced on a HiSeq 2500 Illumina platform to obtain 50-bp single-end reads (TruSeq Rapid kit; Illumina).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
all_counts.tsv
Mtb14_45
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| Data processing |
Reads passing Illumina filters were further filtered for those mapping to tRNA, rRNA, adapters, and spike-in controls using Bowtie v1.0.0 (-S --fr)
Filtered reads were mapped against the human GRCh38 reference using Tophat v1.4.1 (--library-type fr-firststrand)
Reads mapping to the reference genome are further filtered to remove low-complexity reads with PRINSEQ v0.20.3 (-lc_method "dust" -lc_threshold 4)
Sequencing read coverage per gene was counted using HTSeq-count v0.7.1 (-m union -s yes -t exon -i gene_id) and features with 0 counts across all samples were removed
Genome_build: GRCh38
Supplementary_files_format_and_content: count matrix with rows as genes and columns as samples
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| Submission date |
Aug 18, 2020 |
| Last update date |
Dec 06, 2021 |
| Contact name |
Paige Dubelko |
| E-mail(s) |
[email protected]
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| Organization name |
La Jolla Institure for Immunology
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| Lab |
Peters lab
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| Street address |
9420 Athena Circle
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| City |
San Diego |
| State/province |
CA |
| ZIP/Postal code |
92037 |
| Country |
USA |
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| Platform ID |
GPL24676 |
| Series (1) |
| GSE156422 |
Transcriptional signature of BCG vaccination |
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| Relations |
| BioSample |
SAMN15849436 |
| Supplementary data files not provided |
| Raw data are available in SRA |
| Processed data are available on Series record |
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