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| Status |
Public on Feb 15, 2019 |
| Title |
B9: LN2 CD69- RNA-seq |
| Sample type |
SRA |
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| Source name |
CD8+ T cells
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| Organism |
Homo sapiens |
| Characteristics |
passages: FACS purified cells
tissue: mesenteric lymph node
cell type: CD8+ T cells
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| Extracted molecule |
total RNA |
| Extraction protocol |
RNA-seq: 250 cells per subset were sorted using a FACSAriaII (BD Biosciences) directly into lysis buffer provided with the SMART-Seq v4 Ultra Low Input RNA Kit (Takara) and snap frozen.
RNA-seq: RNA-seq libraries were prepared using the SMART-Seq v4 Ultra Low Input RNA Kit (Takara) according to manufacturer’s protocols. Briefly, 3’ SMART-Seq CDS (oligo-dt) Primers were hybridized to the poly(A) tail of all the mRNA and reverse transcribed with the SMARTScribe RT enzyme. cDNA was pre-amplified using SeqAmp DNA polymerase and frozen. Amplified material was purified using Agencourt AMPure XP beads (Beckman) and cDNA quantity was assessed on a Qubit 3.0 (Thermo) and fragment size was evaluated on a 2100 BioAnalyzer (Agilent). The PCR products were next indexed using the Nextera XT DNA Library Prep Kit (Illumina) according to manufacturer’s protocol. Briefly, products were tagmented using the Amplicon tagment mix, containing Tn5 transposase, and indexed using Nextera index 1 (i7) and index 2 (i5) primers. The libraries were again cleaned-up with Agencourt AMPure XP beads, pooled, quantified and sequenced across 75 base pairs (bp) using a paired-end strategy with a 150-cycle high output flow cell on a NextSeq 550 (Illumina).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
NextSeq 550 |
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| Data processing |
RNA-seq: Fastq files from three sequencing runs were concatenated and aligned using STAR 2.5.2a and hg38 to generate a final unique paired mapped read depth ranging from 8 million to 13.6 million reads per sample. The aligned files were normalized using Deseq2.
Genome_build: mm10
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| Submission date |
Feb 15, 2018 |
| Last update date |
Nov 14, 2019 |
| Contact name |
Marcus Buggert |
| E-mail(s) |
marcus.buggert@ki.se
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| Organization name |
Karolinska Institutet
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| Department |
Microbiology
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| Street address |
C2:94; Karolinska Universitetssjukhuset Huddinge
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| City |
Stockholm |
| State/province |
PA |
| ZIP/Postal code |
141 86 |
| Country |
Sweden |
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| Platform ID |
GPL21697 |
| Series (1) |
| GSE110684 |
Identification and characterization of HIV-specific resident memory CD8+ T cells in human lymphoid tissue |
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| Relations |
| BioSample |
SAMN08545559 |
| SRA |
SRX3703072 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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