|
Status |
Public on Mar 22, 2023 |
Title |
CD4_DF4A_4_AB2961 |
Sample type |
SRA |
|
|
Source name |
Lung
|
Organism |
Macaca mulatta |
Characteristics |
tissue: Lung rna sample: AB2961 cell type: CD4 granuloma T cell animal: DF4A granuloma: 4 cfu: 1560 cd4 t-cell_count: 31896
|
Treatment protocol |
Briefly, 3-4 year old animals were housed and maintained in biocontainment racks in accordance with the Animal Welfare Act in a biosafety level 3 (BSL3) vivarium that is accredited by AAALAC International. Primates were bronchoscopically infected with 40-80 CFU of mCherry-reporter Mycobacterium tuberculosis strain Erdman and cared for until termination of study five weeks later. All animal procedures were approved by the National Institute of Allergy and Infectious Disease (NIAID) Division of Intramural Research (DIR) Animal Care and Use Committee under animal study LPD-25E. Other data from the rhesus macaques used in this study appeared in a previously study
|
Extracted molecule |
total RNA |
Extraction protocol |
single cell suspensions of individual pulmonary granulomas were stained with antibodies (clone) CD11b (ICRF44), CD3 (SP34-2), CD4 (SK3), CD8 (RPA-T8), and CD95 (DX2) for 30 minutes at 4°C (Foreman et al., 2016; Kauffman et al., 2021). Samples were washed and stained with Fixable viability dye eFluor780 from eBioscience/ThermoFisher Scientific (Massachusetts, USA) for 20 minutes at 4°C and thoroughly washed using 1X PBS with 1% fetal bovine serum (FBS). Samples were sorted using a BD FACS Aria in a biosafety cabinet inside a BSL3 laboratory. Sorted cell populations were pelleted and stored in Trizol at -80°C until RNA isolation. RNA was isolated using Direct-zol RNA kit from Zymo Research (California, USA) according to the manufacturers protocol. RNA was assessed for quality and degradation using the 2100 Bioanalyzer System from Agilent (Santa Clara, CA), with the product Bioanalyzer RNA 6000 pico assay. Library preparation was completed with the SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing from Takara Bio Inc. (Shiga, Japan) according to manufacturer’s protocol to create full-length cDNA, using an input of 2 ul of total RNA for each sample. Quantification of cDNA was performed using Qubit 2.0 from ThermoFisher Scientific (Waltham, MA) with the Qubit dsDNA HS Assay Kit. The resultant full-length cDNA was used to create final sequencing libraries with the Nextera XT DNA Library Preparation Kit from illumina (San Diego, CA) and the Nextera XT Index Kit v2 Set A from illumina, using 150pg of each cDNA product as input
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|
|
Library strategy |
RNA-Seq |
Library source |
transcriptomic |
Library selection |
cDNA |
Instrument model |
Illumina NextSeq 500 |
|
|
Description |
TPM_all.csv clc_mapping_stats.csv
|
Data processing |
Paired-end sequencing was completed on an illumina NextSeq 500 system, running illumina NextSeq Control Software System Suite version 2.1.0 and RTA version 2.4.11. The final library pool was sequenced via 2 x 76 bp run configuration using the product NextSeq 500/550 High Output Kit v2.5 (150 Cycles From Fastq files, reads were trimmed and mapped and normalized to TPM using CLC Genomics workbench read mapping parameters: Mismatch cost=2;Insertion cost=3;Deletion cost=3;Length fraction=0.8;Similarity fraction=0.8;Global alignment=TRUE;Color space alignment=TRUE;Color error cost=3;Auto-detect paired distances=TRUE;Strand specific=Both;Maximum number of hits for a read=10;Count paired reads as two=FALSE;Expression value=TPM;Calculate expression for genes without transcripts=TRUE;Minimum read count fusion gene table=5; Assembly: GCF_000772875.2_Mmul_8.0.1 Supplementary files format and content: Mapping statistics and raw read counts, per gene, per sample "clc_mapping_stats.csv" Supplementary files format and content: Table of gene expression (TPM) "TPM_all.csv"
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|
|
Submission date |
Mar 19, 2023 |
Last update date |
Mar 22, 2023 |
Contact name |
Timothy G Myers |
E-mail(s) |
tgm@nih.gov
|
Organization name |
National Institute of Allergy and Infectious Diseases
|
Department |
Research Technologies Branch
|
Lab |
Genomic Technologies Section
|
Street address |
50 South Drive, Room 5509
|
City |
Bethesda |
State/province |
MD |
ZIP/Postal code |
20892-8005 |
Country |
USA |
|
|
Platform ID |
GPL21120 |
Series (2) |
GSE227653 |
Gene expression profiling of granuloma T cells in M. tuberculosis infected rhesus macaques reveals a critical role for CD30 [Macaca mulatta] |
GSE228114 |
Gene expression profiling of granuloma T cells in M. tuberculosis infected rhesus macaques reveals a critical role for CD30 |
|
Relations |
BioSample |
SAMN33819063 |
SRA |
SRX19719389 |