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Sample GSM5170844 Query DataSets for GSM5170844
Status Public on Mar 13, 2021
Title Naive post EG7.OVA tumor challenge blood - Mouse 2 RUB157_1_2_Naive_postblood
Sample type SRA
 
Source name Blood
Organism Mus musculus
Characteristics strain: B6.SJL-Ptprc<a> Pepc<b>/BoyJ
treatment: treatment naïve mouse, with OT-1 T cell transfer, 7 days post EG7.OVA rechallenge
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.
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.
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.
 
Library strategy OTHER
Library source genomic
Library selection other
Instrument model Illumina NextSeq 500
 
Description Naive post EG7.OVA tumor challenge blood - Mouse 2
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
 
Submission date Mar 12, 2021
Last update date Mar 13, 2021
Contact name Anna Salzberg
Organization name Rubius Therapeutics
Street address 399 Binney St UNIT 300
City Cambridge
State/province MA
ZIP/Postal code 02139
Country USA
 
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
Relations
BioSample SAMN18288079

Supplementary file Size Download File type/resource
GSM5170844_RUB157_1_2_Naive_postblood.fasta.gz 76.2 Kb (ftp)(http) FASTA
GSM5170844_RUB157_1_2_Naive_postblood.tsv.gz 210.4 Kb (ftp)(http) TSV
Raw data not provided for this record
Processed data provided as supplementary file

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