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| Status |
Public on Jun 04, 2024 |
| Title |
MP, CONTROL, M0010508-1 |
| Sample type |
SRA |
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| Source name |
MP
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| Organism |
Homo sapiens |
| Characteristics |
cell type: MP
tb status: CONTROL
Sex: M
age: 65
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| Growth protocol |
Peripheral blood mononuclear cell (PBMC) samples from patients who had been infected with Mtb and either progressed to active TB disease (cases) or remained TB-disease free until sampling (controls) were separated using standard Ficoll density gradient centrifugation from 50mL of venous blood and cryopreserved at 5e6 cells/vial. PBMC samples were thawed, and CD14-expressing monocytes were magnetically sorted using the Miltenyi pan monocyte isolation kit, humans (Miltenyi) following the manufacturer’s instructions in magnetic-associated cell sorting (MACS) buffer: 0.5% bovine serum albumin, 2mM ethylene-diamine-tetra-acetic acid (EDTA) in 1X phosphate-buffered saline (PBS). To generate either monocyte-derived dendritic cells (DC) or macrophages (MP), monocytes were washed and resuspended in supplemented complete RPMI (5% fetal calf serum, 1mM 2-mercaptoethanol, penicillin-streptomycin, L-Glutamine, HEPES buffer, both essential and non-essential amino acids, and sodium hydroxide). The media was either supplemented with 300 Units/mL of granulocyte-macrophage colony stimulating factor (GM-CSF) and 200 Units/mL of interleukin (IL)-4 to generate monocyte-derived dendritic cells (DCs), or 20ng/mL of macrophage colony stimulating factor (M-CSF) to generate monocyte-derived macrophages (MPs). For both cell types, monocytes were differentiated in 6-well tissue culture plates for 6 days, then split between analytical flow cytometry to test viability and differentiation, and cell sorting to generate pure populations for RNA extraction.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Cells were lysed with TCL buffer (Qiagen).
RNA-seq library preparation was performed at the Broad Technology Labs (Broad Institute) using a modified Smart-seq protocol for low-input RNA-sequencing, which improves detection of full-length transcripts using template switching and pre-amplification. There were 3 plates of 96 samples each including blank samples. Samples from cases and controls and the two cell-types were randomized across all three plates. Sequencing was performed in an Illumina NextSeq500 as paired-end 2 x 25 bp reads with an additional 8 cycles for each index.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
X000008561269_A01
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| Data processing |
Read alignment was performed using STAR (v2.4.2) with the following parameters: -- twopassMode Basic, -- alignIntronMax 1000000, --alignMatesGapMax 1000000, --sjdbScore 2, --quantMode TranscriptomeSAM, --sjdbOverhang 24. RSEM (v1.2.21) was then used for gene quantification with paired-end mode. We used the hg38 reference genome and GENCODE annotation version 24 (Ensembl 83).
Assembly: hg38
Supplementary files format and content: counts.txt.gz is a tab-delimeted file with expected gene counts as calculated by RSEM.
Supplementary files format and content: log2tpm.txt.gz is a tab-delimeted file with the TPM values as calculated by RSEM that were then scaled to log2(tpm+1).
Supplementary files format and content: metadata.txt.gz is a tab-delimeted file with metadata for each sample.
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| Submission date |
Jun 04, 2024 |
| Last update date |
Jun 04, 2024 |
| Contact name |
Maria Gutierrez-Arcelus |
| Organization name |
Boston Children's Hospital
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| Street address |
1 Blackfan Circle
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| City |
Boston |
| ZIP/Postal code |
02115 |
| Country |
USA |
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| Platform ID |
GPL18573 |
| Series (1) |
| GSE269009 |
RNA-seq of monocyte derived macrophages and dendritic cells in a Peruvian Tuberculosis cohort |
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| Supplementary data files not provided |
| Raw data not provided for this record |
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