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| Status |
Public on May 15, 2024 |
| Title |
old 1 |
| Sample type |
SRA |
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| Source name |
bone marrow
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| Organism |
Mus musculus |
| Characteristics |
tissue: bone marrow
cell type: hematopoietic stem cell
cell type: purified hematopoietic stem cell
age: Old
genotype: Gfp-Lc3
treatment: steady state isolation
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| Extracted molecule |
total RNA |
| Extraction protocol |
For 10X Genomics sequencing, ~ 30,000 HSCs were sorted into 1.5 ml tubes containing 500 µl of HBSS/2% FBS and transferred to the Columbia Genome Center Single Cell Analysis Core Facility for microfluidic cell processing, library preparation and sequencing. For SmartSeq2 sequencing, Single yHSC, AThi oHSC, ATmid oHSC, and ATlo oHSC were directly sorted into individual wells of a 96-well PCR plate in 2.3 µl of lysis buffer containing 0.2% Triton X-100 (Sigma-Aldrich) and 1U of Superase-In RNase Inhibitor (Ambion). Cells were frozen immediately at -80°C until further processing.
For 10X, ~10,000 HSCs were loaded for each sample and 1 sample was loaded per condition. For SmartSeq2, after thawing on ice, 2 µl of an annealing mixture containing1 µM oligodT (IDT), 5 mM each dNTPs and a 1:6,000,000 dilution of ERCC RNA Spike-In Mix (Invitrogen) was added followed by incubation at 72°C for 3 min. Then 5.7 µl of a Reverse Transcription mix containing 3.5 U/µl of Maxima H minus retrotranscriptase (ThermoFisher), 0.88 U/µl of Superase-In RNase Inhibitor, 1.75x Maxima RT Buffer, 3.5 µM TSO (Qiagen) and 13.15% PEG 8000 (Sigma-Aldrich) was added and the mixture was incubated at 42°C for 90 min, followed by 70°C for 15 min. cDNA was further amplified by adding 40 µl of a PCR mix containing 0.03 U/µl of Terra PCR direct polymerase (Takara Bio), 1.25x Terra PCR Direct Buffer, and 0.25 µM IS PCR primer (ID). PCR was as follows: 3 min at 98°C for initial denaturation followed by 21 cycles of 15 s at 98°C, 30 s at 65°C, 4 min at 68°C. Final elongation was performed for 10 min at 72°C. Following preamplification, all samples were purified using Ampure XP beads (Beckman Coulter) at a ratio of 1:0.6 with a final elution in 25 µl of EB Buffer (Qiagen). The cDNA was then quantified using the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher). Size distributions were checked on high-sensitivity DNA chips (Agilent Bioanalyzer).
For 10X, samples were sequenced using an Illumina HiSEQ 4000 instrument. For SmartSeq2, samples were used to construct Nextera XT libraries (Illumina) from 100 pg of preamplified cDNA. Libraries were purified and size selected (0.5x-0.7x) using Ampure XP beads. Libraries were quantified using KAPA qPCR quantification kit (KAPA Biosystems), pooled and sequenced using an Illumina HiSEQ 4000 instrument.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic single cell |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
For 10X, QC was performed keeping cells with more than 500 genes detected, less than 100,000 total reads, and less than 20% mitochondrial reads. Downstream analyses were done using Scanpy with cells normalized to 10,000 UMIs per cell and logarithmically transformed. Highly variable genes (HVGs) were selected using the “FindVariableFeatures” method”. UMAP visualizations were obtained from 50 PCA components. Cell clusters were defined using either Louvain or Leiden algorithms.
For SmartSeq2, reads were mapped to the Mus musculus genome (EMSEMBL GRCm38.p4 Release 81) and ERCC sequences using GSNAP (version 2015-09-29) with parameters: -A sam –B 5 –t 24 –n 1 –Q –N 1. HTseq-count56 was used to count reads mapped to each gene, with parameters: –s no. All cells with < 100,000 reads mapping to endogenous RNA and >20% reads mapping to mitochondrial genes were considered low quality and removed from downstream analyses. Data were normalized and highly variable genes were identified as previously described57, using a false discovery rate threshold equal to 0.1 for the chi-squared test.
Assembly: EMSEMBL GRCm38.p4 Release 81
Supplementary files format and content: CSV files containing differential gene expression by cluster are provided.
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| Submission date |
Apr 21, 2023 |
| Last update date |
May 15, 2024 |
| Contact name |
Paul Vincent Dellorusso |
| E-mail(s) |
paul.dellorusso@gmail.com
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| Phone |
5166552491
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| Organization name |
Columbia University Irving Medical Center
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| Department |
Genetics and Development
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| Lab |
Passegue Lab
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| Street address |
701 West 168th Street, 724 Hammer Health Sciences Center
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| City |
New York |
| State/province |
NY |
| ZIP/Postal code |
10032 |
| Country |
USA |
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| Platform ID |
GPL24247 |
| Series (2) |
| GSE229137 |
Autophagy engaging and non-engaging hematopoietic stem cells |
| GSE230281 |
yHSC and oHSC scRNAseq with oHSC AThi ATlo Index Sorting. Assessing the transcriptional changes associated with autophagy engagement in old hematopoietic stem cells at single cell resolution. |
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| Relations |
| BioSample |
SAMN34290074 |
| SRA |
SRX20051567 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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