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| Status |
Public on Feb 17, 2025 |
| Title |
H3WT_hemi__P3 |
| Sample type |
SRA |
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| Source name |
Human tissue GBM
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| Organism |
Homo sapiens |
| Characteristics |
age: 8 days old
tissue: pediatric high grade glioma
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
Smart-seq2 protocol, 10X Genomics protocol
Smart-seq2: Library was performed according to the manufacter’s instructions (single cell 3’ v2 protocol). Briefly: RNA was purified with Agencourt RNAClean. Oligo-dT primed reverse transcription (RT) was performed using Maxima H Minus reverse transcriptase (Life Technologies) and a template-switching oligonucleotide (TSO; Qiagen). For the reverse transcription step, Maxima RNaseH-minus RT (200 U/ml) was used as the RT enzyme at 2U/ml. The RT step was performed at 50 Celsius degree for 90 minutes followed by 85 Celsius degree for 5 minutes. In the PCR pre-amplification step, an ISPCR primer was used at 0.2 mM and PCR was performed for 21 cycles. ). PCR purification was performed using AMpure XP beads at 0.8X. Libraries were enriched with dual indexes using Illumina Nextera XT Library Prep kits
10X Genomics: The isolated single cells (T cells, Myeloid cells, or tumor cells) were loaded into separate channels of a Single Cell Chip A with reverse transcriptase reagent mixture and 50ul gel beads according to the manufacturer’s protocol.Chips were next loaded into the 10X Genomics Chromium Controller for single-cell partitioning, immediately followed by emulsion recovery from the chip and incubated in a Thermocycler for the RT reaction. cDNA isolation and library preparation were completed as per the manufacturer’s protocol. Isolated cDNA was amplified (13 cycles). cDNA was allocated for preparation of a gene expression library or TCR enrichment/library preparation with the Chromium Single Cell V(D)J TCR kit (10X Genomics)
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic single cell |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
fresh single whole cell RNA-seq: SmartSeq2 and 10X Genomics
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| Data processing |
Illumina bcl2fastq 1.5 is used for demultiplexing
RNA-seq sequenced reads were mapped to hg19 using hisat2(2.1.0) with parameters --met 5 -k 10 --mp 1,1 --np 1 --score-min L,0,-0.1 --secondary --no-mixed --no-softclip --no-discordant --rdg 99999999,99999999 --rfg 99999999,99999999 --no-spliced-alignment --seed 12345
RNA-seq TPM were calculated using RSEM (v1.3.0) with parameters --estimate-rspd --paired-end -sam
Supplementary files: tab-delimited text files include TPM values for all cells passing QC (immune and tumor) separately for samples profiled by SmartSeq2 and/or 10X Genomics
tab-delimited text files for metadata for all cells passing QC (tumor and immune) separately for samples profiled by SmartSeq2 and/or 10X Genomics
Assembly: hg19
Supplementary files format and content: Tab-separated count matrix files and metadata files
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| Submission date |
Mar 22, 2023 |
| Last update date |
Feb 17, 2025 |
| Contact name |
Mariella Filbin |
| Organization name |
Dana Farber Cancer Institute
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| Street address |
360 Longwood Ave
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| City |
Boston |
| State/province |
Massachusetts |
| ZIP/Postal code |
02215 |
| Country |
USA |
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| Platform ID |
GPL18573 |
| Series (1) |
| GSE227983 |
Immune and tumor cell landscape in pediatric high grade gliomas |
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| Supplementary data files not provided |
| Raw data not provided for this record |
| Processed data are available on Series record |
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