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| Status |
Public on Jul 15, 2022 |
| Title |
mouse_expt_arms_1_2_3_4_5_gex |
| Sample type |
SRA |
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| Source name |
mixed sample, expt arms 1-5, bone marrow and spleen T-cells
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| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
tissue: bone marrow and spleen
treatment: mixed samples: injected with leukemia cell line (LM138), treated with nilotinib and/or with anti-mouse PDL1
sample type: single cell mRNA
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| Treatment protocol |
For the mouse studies, C57BL/6 mice were injected with 2500 LM138 cells via tail vein. Mice were bled at 14 days post-leukemia challenge, then treated with 75mg/kg nilotinib via oral gavage 5 days/week for 2 weeks. Some mice were treated with 10mg/kg InVivoPlus anti-mouse PDL1 (BioXCell, clone: 10F.9G2), or an isotype control antibody (Rat IgG2b, kappa) via intraperitoneal injection on days 14, 16 and 18 post-leukemia establishment. For the 1-week treatment schedule, mice were treated with nilotinib 5 days/week for 1 week starting at day 14 post leukemia establishment, and with anti-PDL1 or isotype control antibodies. For the CD4 and CD8 T cell depletion studies, mice were treated with 22mg/kg InVivoPlus anti-CD4 (BioXCell, clone: GK1.5) or InVivoPlus anti-CD8 (BioXCell, clone: YTS 169.4) depleting antibodies on days 7, 9 and 11 post-leukemia establishment. For the human studies, leukemia blasts, T cells (CD45+CD3+) and other immune cells (CD45+CD3-CD10-) were isolated from bone marrow biopsy samples from five leukemia patients.
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| Extracted molecule |
total RNA |
| Extraction protocol |
For the mouse studies, CD44+CD4+ T cells from the indicated arms of mice were sorted using a BD FACSAria sorter. Cells from different treatment arms were labeled with hashtag antibodies, as well as CITE-Seq antibodies to CD25, OX40, TIGIT, PD1, Tim3, LAG3 (Biolegend). Sorted CD44+CD4+ T cells were resuspended at 106 ml in 50% FBS in 1×PBS and captured using 10x Genomics Single Cell 5’ Solution. Single cell TCR sequencing was performed using the Chromium Next GEM Single Cell V(D)J Reagent Kits v1.1 (10x Genomics) according to the manufacturer’s instructions. For the human studies, cells were sorted using a BD FACSAria sorter. Ten-thousand cells from each of the three sorted fractions were pooled together and resuspended at 10^6 ml in 50% FBS in 1×PBS before capture using 10x Genomics Single Cell 5’ Solution. Single cell TCR sequencing was performed using the Chromium Next GEM Single Cell V(D)J Reagent Kits v1.1 (10x Genomics).
For 10X Genomics scRNA-seq, we generated three libraries that measure: (1) mRNA transcript expression (GEX), (2) sample/tissue-specific hashtag oligos (HTO) and protein marker levels using antibody derived tags (ADT), and (3) T cell receptor specific mRNA reperitorires. Cells were harvested and stained as described above. The sample was split into three libraries (GEX, HTO/ADT, and TCR). Reverse transcription PCR and library preparation were carried out under the Chromium Single Cell 5’ v1.1 protocol (10X Genomics) per manufacturer’s recommendations. The libraries were sequenced on the NovaSeq 6000 with 2x150 bp paired-end reads (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 |
Mixed samples: T cells derived from the bone marrow or spleen of leukemic mice after immunization or niliotinib/anti-PDL1 drug treatments
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| Data processing |
10X Genomics Cell Ranger (scRNAseq) software was used to demultiplex samples and generate fastq files.
Each gel bead in emulsion (GEM) was identified and all gene specific reads were mapped via cellranger "count" (ver. 5.0.1) using the mouse reference genome (mm10) or human reference genome (GRCh38), or antibody specific tags (HTOs and ADTs). T-cell receptor contigs were recovered by running Cellranger "vdj" software with a reference provided by 10X genomics.
HTO/ADT counts were generated by cellranger count using the specific antibody-oligo tags sequences used in this experiment. Experimental arms or patient samples were classified using HTO counts respectively via GMM-Demux software (ver. 0.2.1.3).
Raw counts data was imported into R (ver 4.0.3) for downstream processing, including normalization, principal components analysis, differential gene expression testing, single cell clustering, etc. primarily using the Seurat R package (ver. 4.0.1).
Genome_build: refdata-gex-mm10-2020-A, refdata-gex-GRCh38-2020-A, refdata-cellranger-vdj-GRCm38-alts-ensembl-5.0.0, refdata-cellranger-vdj-GRCh38-alts-ensembl-5.0.0 (all provided by 10X Genomics)
Supplementary_files_format_and_content: tab-delimited text files include raw or normalized read count values for each sample (GEX, HTO, ADT) or tab-delimited text and json files of T-cell receptor contigs (TCR)
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| Submission date |
Feb 02, 2022 |
| Last update date |
Jul 15, 2022 |
| Contact name |
Todd P Knutson |
| E-mail(s) |
[email protected]
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| Phone |
612-626-8911
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| Organization name |
University of Minnesota
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| Department |
Minnesota Supercomputing Institute
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| Street address |
117 Pleasant St SE
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| City |
Minneapolis |
| State/province |
MN |
| ZIP/Postal code |
55455 |
| Country |
USA |
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| Platform ID |
GPL24247 |
| Series (1) |
| GSE195964 |
Combination therapy with nilotinib and PDL1 blockade reverses CD4 T cell dysfunction and prevents relapse in acute B cell leukemia |
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| Relations |
| BioSample |
SAMN25599239 |
| SRA |
SRX14024163 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5857048_singlecell_mouse_gex_norm_counts.txt.gz |
7.7 Mb |
(ftp)(http) |
TXT |
| GSM5857048_singlecell_mouse_gex_raw_counts.txt.gz |
46.9 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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