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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Oct 01, 2019 |
| Title |
BM4 |
| Sample type |
SRA |
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| Source name |
normal donor's bone marrow cells
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| Organism |
Homo sapiens |
| Characteristics |
individual: normal donor
tissue: bone marrow
cell type: CD34+ cells
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| Treatment protocol |
no treatment
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| Growth protocol |
uncultured
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| Extracted molecule |
total RNA |
| Extraction protocol |
Single-cell RNA sequencing libraries were prepared using Chromium Single Cell 3’ Library & Gel Bead Kit v2 (10XGenomics, cat. no. 120237). 10,000 CD34+ cells from each donor were loaded on a GemCode Single-Cell Instrument (10x Genomics, Pleasanton, CA, USA) to generate single-cell Gel Bead in emulsion (GEMs) respectively. Reverse transcription (RT) was performed according to the manufacturer's protocol (55 °C for 2 h then 85 °C for 5 min). After RT, GEMs were broken and the single-strand cDNA was purified with DynaBeads MyOne Silane Beads and SPRIselect Reagent (0.6 × SPRI). cDNA was amplified by PCR (98 °C for 3 min; 98 °C for 15 s; 67 °C for 20 s; and 72 °C for 1 min) for 14 cycles then 72 °C for 1 min. The amplified cDNA product was cleaned up using SPRIselect beads (0.6 × SPRI). The cDNA was enzymatically fragmented and end-repair, dA-tailing and adaptor ligation were performed. The patient samples were differentially indexed using Chromium i7 sample indexing. The barcoded and indexed libraries were quantified by quantitative PCR (KAPA Biosystems Library Quantification Kit for Illumina platforms, cat. no. KK4824) and normalised. The library pool was sequenced on an Illumina NovaSeq6000 (S2 kits, PE100bp) (Novogene). Approximately 3,000-5,000 cells were captured for each donor and at least 100,000 reads per cell were obtained (QC30 shown in supplemental Table 2).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
*I1_001.fastq.gz: contains the sample index.
*R1_001.fastq.gz: contains the cell barcode (first 16 nt) and UMI (next 10 nt).
*R2_001.fastq.gz: contains the RNA sequence.
Single-cell RNA sequencing libraries were prepared using Chromium Single Cell 3’ Library & Gel Bead Kit v2 (10XGenomics, cat. no. 120237). 10,000 CD34+ cells from each donor were loaded on a GemCode Single-Cell Instrument (10x Genomics, Pleasanton, CA, USA) to generate single-cell Gel Bead in emulsion (GEMs) respectively. Reverse transcription (RT) was performed according to the manufacturer's protocol (55 °C for 2 h then 85 °C for 5 min). After RT, GEMs were broken and the single-strand cDNA was purified with DynaBeads MyOne Silane Beads and SPRIselect Reagent (0.6 × SPRI). cDNA was amplified by PCR (98 °C for 3 min; 98 °C for 15 s; 67 °C for 20 s; and 72 °C for 1 min) for 14 cycles then 72 °C for 1 min. The amplified cDNA product was cleaned up using SPRIselect beads (0.6 × SPRI). The cDNA was enzymatically fragmented and end-repair, dA-tailing and adaptor ligation were performed. The patient samples were differentially indexed using Chromium i7 sample indexing. The barcoded and indexed libraries were quantified by quantitative PCR (KAPA Biosystems Library Quantification Kit for Illumina platforms, cat. no. KK4824) and normalised. The library pool was sequenced on an Illumina NovaSeq6000 (S2 kits, PE100bp) (Novogene). Approximately 3,000-5,000 cells were captured for each donor and at least 100,000 reads per cell were obtained (QC30 shown in supplemental Table 2).
The Cell Ranger Single-Cell Software (1.2.0, 10x Genomics) was used to perform sequencing data demultiplexing, barcode processing and gene counting against the reference genome (GRCh38-1.2.0).
Genome_build: GRCh38-1.2.0
Supplementary_files_format_and_content: tar archive containing genes.tsv, matrix.mtx, barcodes.tsv
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| Submission date |
Jun 22, 2019 |
| Last update date |
Oct 02, 2019 |
| Contact name |
Peng Hua |
| E-mail(s) |
penghua@njmu.edu.cn
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| Organization name |
Nanjing Medical University
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| Department |
State Key Laboratory of Reproductive Medicine and Offspring Health
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| Street address |
101 Longmiandadao
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| City |
Nanjing |
| State/province |
Jiangsu |
| ZIP/Postal code |
211116 |
| Country |
China |
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| Platform ID |
GPL24676 |
| Series (1) |
| GSE133181 |
Single cell transcriptome analysis of normal, Thalassemia and sickle cell donors' bone marrow CD34+ cells |
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| Relations |
| BioSample |
SAMN12111252 |
| SRA |
SRX6261307 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3901488_BM4.tar.gz |
20.2 Mb |
(ftp)(http) |
TAR |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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