 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Jun 23, 2020 |
| Title |
LungMF KO scRNASeq |
| Sample type |
SRA |
| |
|
| Source name |
alveolar macrophage
|
| Organism |
Mus musculus |
| Characteristics |
strain: FVB/NJ backcross to C57BL/6 over 10 generations
tissue: lung
cell type: alveolar macrophage
age: 8-12 weeks
genotype: HDAC3 KO
|
| Extracted molecule |
total RNA |
| Extraction protocol |
The CD45+CD11c+SiglecF+ lung alverolar macrophages (AMs) were sorted by FACS Aria II flow cytometer. 5,000 FACS-sorted cells were loaded into each reaction for gel bead-in-emulsion (GEM) generation and cell barcoding.
Single cell sequencing libraries were generated using the 10X Genomics Chromium Single Cell 3’ Reagent Kit (v2 Chemistry) and Chromium Single Cell Controller. Reverse transcription of the GEM (GEM-RT) was performed in a Thermocycler (Veriti™ 96-Well Fast Thermal Cycler, Applied Biosynthesis; 53 °C 45 min, 85 °C 5 min, 4 °C hold). cDNA amplification was performed after GEM-RT cleanup with Dynabeads MyOne Silane (Thermo Fisher Scientific) with the same Thermocycler (98 °C 3 min; 98 °C 15 sec, 67 °C 20 sec, 72 °C 1 min, repeat 12 cycles; 72 °C 1 min, 4 °C hold). Amplified cDNA is cleaned up with SPRIselect Reagent Kit (Beckman Coulter) and followed by library construction procedure, including fragmentation, end repaired, adaptor ligation, and library amplification. A Bioanalyzer (Agilent) was used for library quality control. Libraries were sequenced on an Illumina HiSeq4000 using paired-end flow cell: Read 1, 26 cycles, i7 index 8 cycles; Read 2: 98 cycles.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 4000 |
| |
|
| Data processing |
Paired-end read counts for all known murine genes underwent barcode and unique molecular identifier (UMI) deconvolution, and were subsequently aligned to the mm10 reference genome using the 10X Cell RangerTM [v2.1] pipeline.
2,000-3,000 cells were captured, with 53,000-73,000 mean reads per cell, with 1,200-1,300 median genes per cell. Datasets were subsequently analyzed using R package Seurat’s Canonical Correlation Analysis (CCA) workflow to characterize the differences between pooled samples.
The following quality control metrics were employed. To identify potential doublets, cells expressing uncharacteristically high numbers of genes (>2,500) were excluded (double the median number of genes). Low quality cells were excluded based on a low number of genes detected (<300) or having high mitochondrial genetic content (>5.0%). Additionally, uninteresting sources of variation within the data were removed. Genes removed include sex specific genes (Xist and all Y chromosome genes), ribosomal structural proteins (as identified by gene ontology term GO:0003735 and RPG: Ribosomal Protein Gene database (41), non-coding rRNAs, Hbb, and genes expressed in < 3 cells.
Samples have 3 fastq files associated with them with a tag in the filename such R1,R1, I1. The I1 contains the sample index. The R1 contains the 10x barcode (16 base pairs) followed by UMI (10 bp). The R2 contains the transcript read.
Genome_build: mm10
Supplementary_files_format_and_content: [LGF_1.data.txt, LGF_2.data.txt] The data slot stores normalized and log-transformed single cell expression. This maintains the relative abundance levels of all genes, and contains only zeros or positive values. This data is used for visualizations, such as violin and feature plots, most differential expression tests, finding high-variance genes, and as input to ScaleData.
[LGF_1.scale.data.txt, LGF_2.scale.data.txt] The scale.data slot represents a cell’s relative expression of each gene, in comparison to all other cells. Therefore this matrix contains both positive and negative values. This data is used as input for dimensional reduction techniques, and is displayed in heatmaps
|
| |
|
| Submission date |
Nov 14, 2018 |
| Last update date |
Jun 26, 2020 |
| Contact name |
Qing-Sheng Mi |
| E-mail(s) |
qmi1@HFHS.ORG
|
| Organization name |
Henry Ford Health System
|
| Department |
Dermatology
|
| Lab |
Immunology Program
|
| Street address |
One Ford Place
|
| City |
Detroit |
| State/province |
Michigan |
| ZIP/Postal code |
48202 |
| Country |
USA |
| |
|
| Platform ID |
GPL21103 |
| Series (2) |
| GSE122529 |
The epigenetic regulator Histone Deacetylase 3 regulates the ontogeny and maintenance of tissue-resident macrophage [scRNA-Seq] |
| GSE122533 |
The epigenetic regulator Histone Deacetylase 3 regulates the ontogeny and maintenance of tissue-resident macrophage |
|
| Relations |
| BioSample |
SAMN10422832 |
| SRA |
SRX5010708 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3473314_LGF_2.data.txt.gz |
35.0 Mb |
(ftp)(http) |
TXT |
| GSM3473314_LGF_2.scale.data.txt.gz |
268.2 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
|
|
|
|
 |
