|
| Status |
Public on Mar 13, 2018 |
| Title |
JK CD63Cap 2 |
| Sample type |
SRA |
| |
|
| Source name |
Jurkat cell line exosomes bound by CD63
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: E6.1
cell type: Jurkat T cell line
tissue source: myeloid
sample type: Exosome bound by CD63
|
| Treatment protocol |
The cultured Jurkat were washed with IXPBs followed by a second wash with HITES medium (DMEM/F12 supplemented with, bovine serum albumin, hydrocortisone, insulin, transferrin, and trace elements). EVs were isolated from Jurkat T cells were cultured overnight using HITES medium. The conditioned media from Jurkat T cells were collected, and cell debris were removed using centrifugation at 300 g for 5 minutes and 2500 g for 10 minutes respectively. Centrifuged media were concentrated using Ultra-4 centrifugal filters to about 1 ml volume and further centrifuged at 10,000 rpm for 10 minutes. EVs were isolated using an Exo-Quick kit (SBI) and CD63, CD47 and MHC1 positive EVs were captured using conjugating MNPs (15nM) with respective antibodies. The uncaptured samples (not bonded) used as Flow samples.
|
| Growth protocol |
The Jurkat T cell line (E6.1) was purchased from ATCC. The cells were maintained using RPMI 1640 containing 10% FBS, glutamine, penicillin, and streptomycin (Gibco). The Jurkat and JinB8 T cells used for experiments were maintained less than 6 weeks in culture.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Trizol (Invitrogen) and RNaseazy (Qiagen) kits were used to extract total RNA according to the manufacturer's instructions.
Small RNA sequencing libraries were size selected (<150bp) using the Agencourt AMPure beads. KAPA qPCR Library Quantification Kit was applied prior to pooling for the final libraries validation and normalization. The sequencing was done with 12.5 million reads per sample with 75 bp reads. (Multiplexing 32 samples on a 400 million read flow cell).
|
| |
|
| Library strategy |
ncRNA-Seq |
| Library source |
transcriptomic |
| Library selection |
size fractionation |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
captured using conjugated MNPs to CD63 Antibody
AB2335-L1_JK_CD63Cap_HLT7JBGXY_S2_R1_001
Slxg-HLT7JBGXY-L1-D702-D504
|
| Data processing |
RNA sequencing reads were aligned by DNASTAR version 14 SeqMan NGen to either small coding/noncoding RNAs (CDS, rRNA, tRNA, ncRNA, tmRNA and miscRNA) or to miRNA reference (mature and precursors) evaluate small RNAs. DNASTAR version 14.1 ArrayStar was used for differential expression analysis.
Genome_build: miRbase 2.1, GRCh38
|
| |
|
| Submission date |
Sep 05, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Sukhbir Kaur |
| E-mail(s) |
kaurs@mail.nih.gov
|
| Organization name |
NIH
|
| Department |
NCI
|
| Lab |
LP
|
| Street address |
10 Center Drive
|
| City |
Bethesda |
| ZIP/Postal code |
20892 |
| Country |
USA |
| |
|
| Platform ID |
GPL18573 |
| Series (1) |
| GSE103493 |
Jurkat cell exosomes bound by CD47, CD63 and MHC1 harbor different RNA content |
|
| Relations |
| BioSample |
SAMN07605615 |
| SRA |
SRX3161491 |
