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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Mar 13, 2021 |
| Title |
Cured pre EG7.OVA tumor challenge blood - Mouse 7 RUB157_5_7_Cured_preblood |
| Sample type |
SRA |
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| Source name |
Blood
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| Organism |
Mus musculus |
| Characteristics |
strain: B6.SJL-Ptprc<a> Pepc<b>/BoyJ
treatment: mRBC-OVA-4-1BBL-IL12 with prior OT-1 T cell transfer cured mouse, 1 day before EG7.OVA rechallenge
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| Treatment protocol |
For paired blood and tumor analyses (RUB170), Pep Boy (CD45.1 B6) mice were injected subcutaneously in the flank with 2×106 EG7.OVA at a 1:1 ratio in Roswell Park Memorial Institute (RPMI) 1640 media and Matrigel® matrix (Corning, PA, USA). Tumor-bearing mice were randomized when the average tumor volume reached ~175 mm3 and 1×106 naive OT-1 cells were administered to all groups no later than one day after randomization by tail vein injection. Several hours after T cell injection, mice were treated intravenously 1×109 mRBC-CTRL or a dose titration of mRBC-OVA-4-1BBL-IL-12 (1×109, 2.5×108) in 200 μL PBS on days 0 and 3. For blood analyses before and after tumor re-challenge (RUB157), CD45.1 Pep Boy mice were randomized when EG7.OVA tumors (2×106 injected) reached ~230 mm3, treated with 1×106 naïve OT-1 cells, and dosed with 2.5×108 mRBC-OVA-4-1BBL-IL-12. Mice cured of original EG7.OVA tumors (n=7) were rechallenged on day 66 with EG7.OVA. Age-matched naïve CD45.1 Pep Boy mice (n=5) were treated on day 65 with 5×105 naïve OT-1 cells one day before challenge with EG7.OVA cells, as controls. All previously cured mice rejected EG7.OVA rechallenge. At 61 days post-second EG7.OVA challenge on day 127, cured mice (n=7) along with age-matched naïve control mice (n=5) were challenged with EL4.
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| Growth protocol |
Murine lymphoma cell lines EL4 (TIB-39™) and EG7.OVA (CRL-2113™) were obtained from American Type Culture Collection (ATCC®) and cultured as recommended by ATCC.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Blood was collected by submandibular bleed or cardiac puncture in BD Microtainer® blood collection tubes with K2EDTA (BD Biosciences) and the tubes were kept on ice until analysis. Genomic DNA (gDNA) was extracted from 50‒60 μL of anticoagulated blood using DNeasy® Blood and Tissue Kit (Qiagen® Inc.) according to the manufacturer’s instructions. Tumors were digested in gentleMACS™ C‑tubes (Miltenyi Biotec, Inc.) using the murine Tumor Dissociation Kit. Cell suspensions were generated using a gentleMACS Octo Dissociator with Heaters instrument (Miltenyi Biotec, Inc.) according to the manufacturer’s instructions. Digested tissue was then filtered through a 70 µm cell strainer and washed. gDNA was extracted from one quarter of the tumor single cell suspensions using the DNeasy Blood and Tissue Kit (Qiagen Inc.) according to the manufacturer’s instructions.
Immunosequencing of the CDR3 regions of mouse TCRβ chains was performed using the ImmunoSEQÒ platform (Adaptive Biotechnologies Corporation). In brief, extracted DNA was amplified in a bias-controlled multiplex polymerase chain reaction (PCR), followed by high‑throughput sequencing. Sequences were then collapsed and filtered in order to identify and quantitate the absolute abundance of each unique TCRβ CDR3 region for further analysis as previously described.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
Cured pre EG7.OVA tumor challenge blood - Mouse 7
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| Data processing |
Raw data were processed using the standard immunoSEQ Analyzer from Adaptive Biotechnologies Corporation.
*** Raw fastq files not provided by third party provider ***
*** Third party provider only releases processed data which consist of nucleotide sequences generated through V(D)J recombination as well as associated information including amino acid translation and V, D and J gene and allele identifications.***
Genome_build: Third party provider description: "Sequences were not aligned against a reference genome; instead they were aligned and annotated based on our custom pipeline using primers designed to IMGT reference V and J genes. The CDR3 regions were defined as per IMGT beginning with the second conserved cysteine encoded by 3’ portion of V gene segment. IMGT reference: Yousfi Monod, M., Giudicelli, V., Chaume, D. & Lefranc, M. P. IMGT/JunctionAnalysis: the first tool for the analysis of the immunoglobulin and T cell receptor complex V-J and V-D-J JUNCTIONs. Bioinformatics 20, (Suppl 1): i379–i385 (2004).
Supplementary_files_format_and_content: TCRbeta rearrangement text files
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| Submission date |
Mar 12, 2021 |
| Last update date |
Mar 13, 2021 |
| Contact name |
Anna Salzberg |
| Organization name |
Rubius Therapeutics
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| Street address |
399 Binney St UNIT 300
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| City |
Cambridge |
| State/province |
MA |
| ZIP/Postal code |
02139 |
| Country |
USA |
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| Platform ID |
GPL19057 |
| Series (1) |
| GSE168826 |
TCR beta sequencing of blood and tumor samples from EG7.OVA bearing wildtype mice with OT-1 T cell transfer |
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| Relations |
| BioSample |
SAMN18288092 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5170854_RUB157_5_7_Cured_preblood.fasta.gz |
146.6 Kb |
(ftp)(http) |
FASTA |
| GSM5170854_RUB157_5_7_Cured_preblood.tsv.gz |
412.1 Kb |
(ftp)(http) |
TSV |
| Raw data not provided for this record |
| Processed data provided as supplementary file |
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