|
| Status |
Public on Jun 16, 2022 |
| Title |
Xenopus_post-treat_only_2 |
| Sample type |
RNA |
| |
|
| Source name |
Xenopus, post-infection treatment only
|
| Organism |
Xenopus laevis |
| Characteristics |
infection: Y
treated post-infection: Y
embryo prophylaxis: N
pathogen prophylaxis: N
|
| Treatment protocol |
Bacteria were streaked out from frozen glycerol stock of target bacterial strain on sheep blood agar media plates overnight at 37°C. A single colony was selected from the streak and grown in LB medium at 37°C. Exponentially growing bacteria were pelleted and resuspended in sterile saline/dextrose with 15% glycerol at a concentration of 1X109 CFU/ml. Faber-Nieuwkoop stage 13/14 embryos were injected with fresh bacterial suspensions using borosilicate glass needles calibrated for a bubble pressure of 25–30 kDa and 0.4 sec pulses to deliver 103–104 CFU of bacteria to each embryo. Bacterial injections of A. Hydrophila were performed with embryos submerged in 3% Ficoll prepared in 0.1X MMR. Prophylaxis and/or post-infection treatment of 40uM 1,4-DPCA was delivered to Xenopus and samples were collected 24h post-infection.
|
| Growth protocol |
Xenopus laevis embryos were fertilized in vitro according to standard protocols in 0.1X Marc’s Modified Ringer’s solution (MMR; 10 mM Na+, 0.2 mM K+, 10.5 mM Cl–, 0.2 mM Ca2+, pH 7.8) and housed at 18°C. Since exclusively sterile filtered solutions, including MMR culture medium, were used to avoid confounding effects of seasonally-varying environmental bacteria, we dosed embryos overnight with 2.64 ul KoiZyme probiotic solution (Koi Care Kennel, Las Vegas, NV) per 1L of 0.1% MMR to avoid dysbiosis.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
For each experiment, RNA from each well containing 10 Xenopus embryos (n=2-3 replicates/condition) was separately extracted and purified using the RNeasy Micro Kit (Qiagen; Venlo, Netherlands).
|
| Label |
biotin
|
| Label protocol |
RNA samples were processed using a Nugen Ovation PICO WTA System V2 kit. The resulting cDNAs were purified using a Qiagen MinElute PCR Purification Kit following the modifications outlined in the Nugen protocol. The cDNAs were fragmented and labeled using a Nugen Encore Biotin Module.
|
| |
|
| Hybridization protocol |
Hybridization solutions were prepared by combining the fragmented, biotin-labeled cDNAs with hybridization cocktail (Affymetrix Hybridization, Wash, and Stain Kit). The mixtures were incubated in a thermal cycler at 99 °C for 2 min followed by 45 °C for 5 min. then loaded on Xenopus laevis Genome 2.0 arrays and incubated for 16-20 hours at 45 ºC and 60 rpm in an Affymetrix Hybridization Oven 645.
|
| Scan protocol |
Following hybridization, arrays were washed and stained on Affymetrix Fluidics Station 450s using the Affymetrix FS450_0001 protocol with the stains and buffers supplied in the Affymetrix Hybridization, Wash, and Stain Kit. The stained arrays were scanned at 532 nm using an Affymetrix GeneChip Scanner 3000.
|
| Description |
Experiment 2: 1,4 DPCA treatment
|
| Data processing |
Microarray data were extracted from CEL files and Robust Multi-array Average (RMA) normalized in Matlab (Mathworks; Natick, MA) and output with linear scaling.
|
| |
|
| Submission date |
Feb 09, 2022 |
| Last update date |
Jun 16, 2022 |
| Contact name |
Megan M Sperry |
| Organization name |
Wyss Institute at Harvard University
|
| Street address |
3 Blackfan Circle
|
| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02115-5713 |
| Country |
USA |
| |
|
| Platform ID |
GPL10756 |
| Series (2) |
| GSE196424 |
Enhancers of host immune tolerance to bacterial infection discovered using linked computational and experimental approaches II |
| GSE196425 |
Enhancers of host immune tolerance to bacterial infection discovered using linked computational and experimental approaches |
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