|
| Status |
Public on Mar 20, 2024 |
| Title |
mouse splenocytes isotype rep5 |
| Sample type |
RNA |
| |
|
| Source name |
CD45pos_IgG_Day5
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
time point: Day5
treatment: IgG
|
| Treatment protocol |
Female C57BL/6 mice were purchased (~6 weeks old) from Taconic labs. Mice were infected i.p. with 20 cysts of Me49 strain of T. gondii . Anti-IL-27p28 antibody was injected i.p. (1mg/mouse) at day 0, 4, 7, 10 or with same dose of isotype IgG
|
| Growth protocol |
Mouse splenocytes were prepared by mechanical dissociation of whole spleens, followed by ACK lysis of red blood cells. After staining with antibody cocktails, total CD45+ cells (day 5; n=5 per treatment) and tetramer positive T-cells (day 10; n=5 per treatment) were sorted by FACS directly into lysis buffer.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from sorted cells with the RNeasy® Mini Kit (Qiagen, Cat. No: 74104)
|
| Label |
biotin
|
| Label protocol |
All procedures were performed as described in the GeneChip® Whole Transcript (WT) Plus Reagent Kit Manual (Affymetrix, Santa Clara, CA). Briefly, the total RNA was isolated using an RNeasy kit (Qiagen), and the sample integrity was verified using RNA 6000 Pico Assay RNA chips run in Agilent 2100 Bioanalyzer (Agilent Technologies). The total RNA (200 ng) was reverse transcribed using GeneChip® WT PLUS Reagent Kit (Affymetrix). The obtained cDNA was used as a template for in vitro transcription using GeneChip® WT Expression Kit (Life Technologies). The obtained antisense cRNA was purified using Nucleic Acid Binding Beads (GeneChip® WT PLUS Reagent Kit, Affymetrix) and used as a template for reverse transcription to produce single-stranded DNA in the sense orientation. During this step, dUTP was incorporated. The DNA was then fragmented using uracil DNA glycosylase (UDG) and apurinic/apyrimidinic endonuclease 1 (APE 1) and labeled with DNA labeling reagent covalently linked to biotin using terminal deoxynucleotidyl transferase (TdT, GeneChip® WT PLUS Reagent Kit, Affymetrix).
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| |
|
| Hybridization protocol |
The labeled fragmented DNA was hybridized to the Gene Arrays 2.0ST for 16–18 h in GeneChip® Hybridization oven 640 at 45 °C with rotation (60 r.p.m.). The hybridized samples were washed and stained using Affymetrix fluidics station 450 as per manufacturer’s instruction (Hybridization, Washing and Staining kit, Affymetrix).
|
| Scan protocol |
Microarrays were scanned using Affymetrix GeneArray Scanner 3000 7G Plus (Affymetrix).
|
| Data processing |
Data were processed with RMA expressing (GC-RMA) using brainarray cdfs
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| |
|
| Submission date |
Sep 17, 2021 |
| Last update date |
Mar 20, 2024 |
| Contact name |
Jonathan Hill |
| Organization name |
Surface Oncology
|
| Department |
Immunobiology
|
| Street address |
50 Hampshire St 8th floor
|
| City |
Cambridge |
| State/province |
MA |
| ZIP/Postal code |
02139 |
| Country |
USA |
| |
|
| Platform ID |
GPL16570 |
| Series (1) |
| GSE184350 |
Impact of endogenous IL-27 on innate and adaptive responses during acute and chronic toxoplasmosis |
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