|
| Status |
Public on Nov 29, 2023 |
| Title |
HSPCs, mRNA-derived cDNA |
| Sample type |
SRA |
| |
|
| Source name |
Bone Marrow
|
| Organism |
Mus musculus |
| Characteristics |
tissue: Bone Marrow
cell type: Hematopoietic Stem and Progenitor Cells
treatment: Different cell isolation protocols
genotype: wild type
library type: mRNA
antibodies/tags: none
|
| Treatment protocol |
Bone marrow cells were isolated and cKit-enriched in ice-cold staining buffer (PBS/2%FBS) with or without 5 uM triptolide (TP). Aliquots of cells were next incubated on ice or at 37ºC in culture media (DMEM/10mM HEPES/2%FBS) with or without 5 uM TP for 90 min. Following incubation, the cells were stained with fluorochrome- and oligo-conjugated antibodies and subjected to cell sorting.
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
Lineage-Sca1+cKit+ cells were FACS-purified, counted and checked for viability using hematocytometer. Cell suspension was adjusted to a concentration of 1,250 cells/µL and 54,000 cells were loaded onto Chromium Controller (10x Genomics).
Libraries were generated using the Chromium Next GEM Single Cell 3’ Reagent Kit v3.1 (10x Genomics). mRNA and hashing oligos (HTO) were reverse transcribed, barcoded and purified using a Dynabeads bead cleanup mix. The cDNA was amplified by PCR according to the Chromium Next GEM Single Cell 3’ Reagent Kit v3.1 protocol with additional primer amplifying the HTO-derived tags (5’-GTGACTGGAGTTCAGACGTGTGCTC-3’). SPRI size selection was performed to separate fragments containing mRNA-derived (>300 bp) and HTO-derived (<180 bp) cDNA. mRNA-derived cDNA sequencing library was next generated according to manufacturer’s instructions (10x Genomics). HTO-derived sequencing library was prepared according to CITE-seq_and_Hashing_protocol_190213 (https://cite-seq.com/protocols). HTO-derived fragments we purified with 2X SPRI beads (Beckman Coulter) and amplified using the KAPA HiFI HotStart Library Amplification Kit (Roche) with SI-PCR primer (5’-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTC-3’) as i5 primer (Illumina), TruSeq D708_s primer 5’-CAAGCAGAAGACGGCATACGAGATGCGCATTAGTGACTGGAGTTCAGACGTGTGC-3’ as i7 primer (Illumina) and the following cycling conditions: 95°C for 3 min followed by 12 cycles 95°C for 20 sec; 64°C for 30 sec; 72°C for 20 sec and final elongation 72°C for 5 min. Final cDNA- and HTO-derived libraries were pooled and sequenced on Illumina NovaSeq 6000 instrument using S2 Reagent Kit v1.5 (100 cycles). Preparation of libraries and sequencing were done at the Center for Translational Genomics, Lund University.
|
| |
|
| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Description |
3' mRNA library: read1 file contains cell barcode and UMI; read2 file contains transcript
poly A RNA
|
| Data processing |
Post-sequencing libraries were processed using Cell Ranger 7.0.0. The FASTQ files were aligned to the mouse reference genome (mm10) to create unique molecular identifier (UMI) count tables of gene expression. The HTO demultiplexing was performed with cellhashr, available at [https://github.com/BimberLab/cellhashR]. The following HTO tags were used: ice_noTP/TotalSeq-A0301; 37ºC_noTP/TotalSeq-A0302; ice_TP/TotalSeq-A0303; 37ºC_TP/TotalSeq-A0304.
Further data processing was done with Seurat v4.1.1 (cell and gene filtering, dimensionality reduction, analysis of IER signature).
All code, software versions (conda/mamba environment specifications) etc needed to reproduce the CITE-seq analysis is hosted at [https://github.com/razofz/DB_AKC_citeseq]
Assembly: mm10
Supplementary files format and content: Tab-separated values files and matrix files
Supplementary files format and content: Hdf5 file with filtered count matrix
Supplementary files format and content: Seurat (R package) object, in .rds (Rdata) format
Supplementary files format and content: Comma-separated files for Seurat-processed metadata and UMAP embeddings per cell
Library strategy: CITE-seq (Cellular Indexing of Transcriptomes and Epitopes by sequencing)
|
| |
|
| Submission date |
Feb 06, 2023 |
| Last update date |
Nov 29, 2023 |
| Contact name |
Anna Konturek-Ciesla |
| E-mail(s) |
anna.konturek-ciesla@med.lu.se
|
| Organization name |
Lund University
|
| Department |
Department of Molecular Hematology
|
| Lab |
David Bryder lab
|
| Street address |
Box 188
|
| City |
Lund |
| ZIP/Postal code |
22100 |
| Country |
Sweden |
| |
|
| Platform ID |
GPL24247 |
| Series (1) |
| GSE224590 |
An Erratic Gene Expression Signature in Primary Hematopoietic Stem Cells: Implications for Interpretations of Aging |
|
| Relations |
| BioSample |
SAMN33129421 |
| SRA |
SRX19289670 |
