|
| Status |
Public on Apr 06, 2020 |
| Title |
Gfi1+/- 10x CITE-Seq_mRNA |
| Sample type |
SRA |
| |
|
| Source name |
Bone marrow
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
cell type: Bone marrow derived Modified GMP
genotype: Gfi1+/-
cdna synthesis: 10x Genomics single cell 3’ v2 assay protocol
library prep: 10x Genomics single cell 3’ v2 assay protocol
sequencing parameters: 429 million reads
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Bone marrow was extracted by crushing hindlimbs isolated from mice immediately following euthanasia. The single cell suspension was enriched using CD117 Microbeads on an AutoMACS Pro (Mitenyi) and stained with TotalSeq antibodies and fluorchome-conjugated antibodies for FACS-sorting, then 10,000 cells per sample were loaded in each lane of a Chromium Controller Instrument (10x Genomics).
The 10x Genomics single cell 3’ v2 assay protocol was followed as described until the cDNA amplification step. The cDNA amplification mix was modified to include 1 mL of a 2 mM dilution of ADT PCR additive primer (5’-CCTTGGCACCCGAGAATT*C*C-3’ where * indicates a phosphorotioate bond) and 1 mL of a 2 mM dilution of HTO PCR additive primer (5’-GTGACTGGAGTTCAGACGTGTGC*T*C-3’ where * indicates a phosphorotioate bond) when applicable, replacing 1-2 mL of water. An 0.6X SPRI selection was performed, and the resulting supernatant was set aside (contains ADTs and HTOs) while the beads (mRNA-derived cDNA) were processed according to the standard 10x Genomics single cell 3’ v2 assay protocol. The ADTs and HTOs were further processed according to the CITE-Seq protocol (New York Genome Center, https://cite-seq.com/protocol/). The final pooled library was comprised of 90% mRNA-derived library and 10% ADT library or 85% mRNA-derived library, 10% ADT library, and 5% HTO library prior to sequencing.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 4000 |
| |
|
| Description |
Male GMP mRNA
clusters.Gfi1-Het.txt
RNA.Gfi1-Het.h5
|
| Data processing |
The 10x Genomics libraries were each sequenced to a depth of 373 and 455-million reads, respectively. The 10x Genomics libraries were aligned to the mouse genome (mm10), with unique molecular index quantified (UMI) gene counts obtained using the Cell Ranger workflow (v2.1.0), from the automated filtered cell barcode output. Using the default filtered cellular barcodes, the associated sparse-filtered matrix files were processed in AltAnalyze and ICGS as previously described to obtain normalized gene counts (counts per ten thousands UMIs) and initial predicted cell populations (PMID: 29183737).
The antibody derived tag (ADT)-linked library for each 10x Genomics capture was sequenced with the same 10x Genomics sample. To obtain ADT UMIs for each cellular barcode, each ADT barcode was supplied as input in the CITE-Seq-Counts python program with the Undetermined fastqs files. Prior to visualization in the software SeqGeq (FlowJo Inc.), normalization was performed on the ADT counts using CLR transformation in Excel using the equation: LN(([adt count value]+1)/mean([all adt count values for antibody])).
Genome_build: mm10
Supplementary_files_format_and_content: Tab-delimited text file and HDF5
Supplementary_files_format_and_content: clusters.Gfi1-WT.txt: Cell Clusters: cellHarmony assigned clusters for GMP-P and Ly6g high sorted Gfi1 WT cells
Supplementary_files_format_and_content: clusters.Gfi1-Het.txt: Cell Clusters: cellHarmony assigned clusters for GMP-P and Ly6g high sorted Gfi1+/- cells
Supplementary_files_format_and_content: clusters.Gfi1-K403R-Het.txt: Cell Clusters: cellHarmony assigned clusters for GMP-P and Ly6g high sorted Gfi1-K403R-Het cells
Supplementary_files_format_and_content: clusters.Gfi1-R412X-Het.txt: Cell Clusters: cellHarmony assigned clusters for GMP-P and Ly6g high sorted Gfi1-R412X-Het cells
Supplementary_files_format_and_content: clusters.Gfi1-R412X-Irf8.txt: Cell Clusters: cellHarmony assigned clusters for GMP-P and Ly6g high sorted Gfi1-R412X-R412X cells
Supplementary_files_format_and_content: clusters.Gfi1-R412X-R412X.h5.txt: Cell Clusters: cellHarmony assigned clusters for GMP-P and Ly6g high sorted Gfi1-R412X Irf8+/- cells
Supplementary_files_format_and_content: RNA.Gfi1-WT.h5: RNA: mRNA UMI counts for GMP-P and Ly6g high sorted Gfi1 WT cells
Supplementary_files_format_and_content: RNA.Gfi1-Het.h5: RNA: mRNA UMI counts for GMP-P and Ly6g high sorted Gfi1+/- cells
Supplementary_files_format_and_content: RNA.Gfi1-K403R-Het.h5: RNA: mRNA UMI counts for GMP-P and Ly6g high sorted Gfi1-K403R-Het cells
Supplementary_files_format_and_content: RNA.Gfi1-R412X-Het.h5: RNA: mRNA UMI counts for GMP-P and Ly6g high sorted Gfi1-R412X-Het cells
Supplementary_files_format_and_content: RNA.Gfi1-R412X-Irf8.h5: RNA: mRNA UMI counts for GMP-P and Ly6g high sorted Gfi1-R412X-R412X cells
Supplementary_files_format_and_content: RNA.Gfi1-R412X-R412X.h5: RNA: mRNA UMI counts for GMP-P and Ly6g high sorted Gfi1-R412X Irf8+/- cells
Supplementary_files_format_and_content: ADT.Gfi1-WT.txt: ADT: Antibody UMI counts for GMP-P and Ly6g high sorted Gfi1 WT cells
Supplementary_files_format_and_content: ADT.Gfi1-Het.txt: ADT: Antibody UMI counts for GMP-P and Ly6g high sorted Gfi1+/- cells
Supplementary_files_format_and_content: ADT.Gfi1-K403R-Het.txt: ADT: Antibody UMI counts for GMP-P and Ly6g high sorted Gfi1-K403R-Het cells
Supplementary_files_format_and_content: ADT.Gfi1-R412X-Het.txt: ADT: Antibody UMI counts for GMP-P and Ly6g high sorted Gfi1-R412X-Het cells
Supplementary_files_format_and_content: ADT.Gfi1-R412X-Irf8.txt: ADT: Antibody UMI counts for GMP-P and Ly6g high sorted Gfi1-R412X-R412X cells
Supplementary_files_format_and_content: ADT.Gfi1-R412X-R412X.txt: ADT: Antibody UMI counts for GMP-P and Ly6g high sorted Gfi1-R412X Irf8+/- cells
|
| |
|
| Submission date |
Dec 19, 2019 |
| Last update date |
Apr 06, 2020 |
| Contact name |
H. Leighton Grimes |
| E-mail(s) |
Lee.Grimes@cchmc.org
|
| Phone |
513-636-6089
|
| Organization name |
Cincinnati Childrens Hospital Medical Center
|
| Department |
Immunobiology
|
| Lab |
Grimes
|
| Street address |
3333 Burnet Ave. MLC 7038
|
| City |
Cincinnati |
| State/province |
OH |
| ZIP/Postal code |
45229 |
| Country |
USA |
| |
|
| Platform ID |
GPL21103 |
| Series (2) |
| GSE120409 |
A neutropenia-associated transcription factor mutation differentially impacts target genes in the cell-states traversed during granulocyte specification and commitment |
| GSE142341 |
Mouse models of neutropenia reveal progenitor-stage-specific defects [CITE-Seq] |
|
| Relations |
| BioSample |
SAMN13633916 |
| SRA |
SRX7416706 |
