|
| Status |
Public on May 05, 2024 |
| Title |
Pt009_PBMC |
| Sample type |
SRA |
| |
|
| Source name |
Blood
|
| Organism |
Homo sapiens |
| Characteristics |
alt sample_name: Pt009_PBMC
patient: Pt009
tissue: Blood
cell type: PBMCs
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Peripheral blood samples from pediatric patients with B-ALL receiving Trisangeleucel were collected under the Boston Children’s Hospital and Dana Farber Cancer Institute approved clinical protocol, ‘PREDICT’ (NCT03369353). Patient blood was obtained and PBMCs were isolated by Ficoll-PaquePLUS (GE Healthcare Bio-Sciences) gradient centrifugation. PBMCs were cryopreserved and stored for further use. CD19 CAR-T cells from patient samples were stained with an anti-CD19 CAR antibody (ACRO Biosystem, Cat#: CD9-HF251-25ug). CD3 flow cytometric antibodies were not utilized, to avoid in vitro stimulation, which can result in alteration of the transcriptional profile. Live CARpos and CARneg, CD4/CD8pos and CD20/CD14neg cells were flow cytometrically sorted and immediately processed for single cell sequencing.
All products used in the preparation of these samples are available from 10x Genomics. Samples in this publication were prepared using the Next GEM 5’ v2 Gel Beads with the i7 Multiplex Plate (Single Index) or the Next GEM Single Cell 5’ v2 Sample Index Plate TT (Dual Index). GEMs were generated using v2 Gel Beads and the v1 Target Enrichment kit utilizing the off-the-shelf human/mouse T Cell Mix 1 and 2 premixed primers for the patient samples.
scRNA-Seq and scTCR-Seq
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic single cell |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Data processing |
Paired GEX and VDJ libraries were aligned using the ‘Cellranger multi’ pipeline (v7.0.0) with 32 CPU’s and 128 GB of RAM, with VDJ libraries listed as “vdj-t” to indicate T cell libraries. Samples were aggregated with the ‘Cellranger aggr’ pipeline (v7.0.0) with arguments ‘--normalize=none --nosecondary’. Our reference transcriptome was created with the Cellranger makeref command, and used Homo_sapiens.GRCh38 as the reference genome and Ensembl v107 as the reference for humans.
Assembly: Homo_sapiens.GRCh38
Supplementary files format and content: h5 matrix of single-cell RNA-Seq
Supplementary files format and content: Csv file of clonotype information for single-cell TCR-Seq
|
| |
|
| Submission date |
May 02, 2024 |
| Last update date |
May 05, 2024 |
| Contact name |
Jim Kaminski |
| E-mail(s) |
James.Kaminski@childrens.harvard.edu
|
| Organization name |
Boston Children's Hospital
|
| Street address |
1 Blackfan Circle
|
| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02115 |
| Country |
USA |
| |
|
| Platform ID |
GPL24676 |
| Series (1) |
| GSE266486 |
B cell directed CAR-T cell therapy results in activation of CD8+ cytotoxic CAR-negative bystander T cells in both non-human primates and patients (human) |
|
