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GEO help: Mouse over screen elements for information. |
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| Status |
Public on May 21, 2021 |
| Title |
A5738 |
| Sample type |
SRA |
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| Source name |
Mammary Fat Pad
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| Organism |
Mus musculus |
| Characteristics |
cell syngeneic orthotopic transplant: Mammary fat pad adipose tissue (uninjected, contralateral) in E0771-luciferase breast cancer bearing mice
mouse strain: C57BL/6J
lean or obese & diet exposure: Formely Obese (high fat diet, HFD, 60% kcal from fat)
surgery: HFD-VSG
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| Treatment protocol |
Female mice were purchased at 3 weeks of age from Jackson Labs C57BL/6J (Jackson stock number: 000664), shipped to UTHSC, acclimated 1 week, then randomized to diets: high fat diet (HFD, D12492i – 60% kcal derived from fat from Research Diets Inc., New Brunswick, NJ) or low fat diet (LFD, D12450Ji- 10% kcal derived from fat) from Research Diets Inc. (New Brunswick, NJ) for 16 weeks (age 4 weeks to 20 weeks old) before a 5 week surgery and cancer protocol..
Perioperative procedures were performed in accordance with the literature. Pre-operative measures included providing liquid diet (Ensure® Original Milk Chocolate Nutrition Shake, Abott) and DietGel recovery (Clear H2O, Portland, ME, ID# 72-06-5022) one day before surgery to all mice. Four hours before surgery, solid food was removed to reduce stomach contents. For 4 hours pre-surgery, mice were maintained half on half off a heat pad in clean cages. Surgery was performed under isoflurane anesthesia. Vertical sleeve gastrectomy (VSG) was performed as previously described. The stomach was clamped and the lateral 80% of the stomach was removed with scissors. The remaining stomach was sutured with 8-0 to create a tubular gastric sleeve. All treatment groups not receiving VSG had a sham surgery performed. For sham, an abdominal laparotomy was performed with exteriorization of the stomach. Light pressure with forceps was applied to the exteriorized stomach. For both VSG and sham surgeries, the abdominal wall was closed with 6-0 sutures and skin closed with staples. Mice received carprofen (5mg/kg, subcutaneous, once daily) as an analgesic immediately prior to and once daily for 3 days following surgery. Mice were given 1ml saline at time of surgery. For 12 hours post-surgery, mice were maintained half on half off a recovery heat pad. Mice were provided Ensure® liquid diet (as above), DietGel recovery, and solid food pellets ad libitum for 48 hours post-surgery. HFD-fed DIO mice receiving VSG (“HFD-VSG”) were maintained on the same HFD for 5 weeks following surgery until euthanasia at study endpoint. Control groups that were lean (“LFD-Sham”) or DIO (“HFD-Sham”) were maintained on respective LFD or HFD diets following sham surgery. For dietary intervention weight loss, “Diet Switch” (DS) mice were formerly HFD-fed DIO switched to LFD ad libitum after sham similar to our previous work (8, 9). “Calorie restricted” (CR) mice were matched controls to the HFD-VSG mice by weight matching through restricting intake of HFD. On average, mice consumed 1.7g (ranging from 1.0-2.5 g or 8.84 kcal (5.2-13.0 kCal) per day of HFD. Mice were fed at the start of the dark cycle.
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| Growth protocol |
E0771 breast cancer cells were thawed from a 1 million cell aliquot stored in liquid nitrogen, plated and passed the same time for every injection. E0771 cells were injected in the left fourth mammary fat pad of 22-week-old C57BL/6J females at 250,000 cells in 100μl of 75% RPMI / 25% Matrigel.
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| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted from uninjected mammary fat pad (contralateral to E0771 injected 4th mammary gland) tissue using kit specific for lipid rich tissue (Norgen Biotek, Ontario, Canada). The integrity of RNA was assessed using Agilent Bioanalyzer and samples with RIN >8.0 were used.
Libraries were constructed using NEBNext® Ultra™ RNA Library Prep Kits (non-directional) for Illumina, following manufacturer protocols. mRNA was enriched using oligo-dT beads.
Libraries were sequenced on NovaSeq 6000 using paired-end 150 bp reads. There was no PhiX spike-in.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
Diet induced obese mice fed HFD, then receiving bariatric surgery vertical sleeve gastrectomy (VSG)
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| Data processing |
RNA degradation was examined by SCISSOR (shape changes in selecting sample outliers in RNA-seq) which uses unsupervised screening of a range of structural alterations in RNA-seq data.
Benjamini-Hochberg procedure was used to control false discovery rate (FDR) for adjusted P value.
Transcript-level abundance was imported into gene-level abundance with the R package tximport.
Genes with low expression were identified and filtered out from further analysis using filterByExpr function of the edgeR package in R software.
Voom transformation function was applied to normalize log2-cpm values using mean-variance trend in the limma software package.
ClaNC was used to create classifier genes that characterize the groups of interest for heatmap and tables.
Database for Annotation, Visualization and Integrated Discovery (DAVID ) v6.8 was used for pathway analysis.
Immune infiltration estimations based on bulk gene expression data from RNA-seq was plotted using TIMER2.0.
Genome_build: GRCm38
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| Submission date |
May 20, 2021 |
| Last update date |
May 21, 2021 |
| Contact name |
Liza Makowski |
| E-mail(s) |
liza.makowski@uthsc.edu
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| Organization name |
UTHSC
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| Department |
Medicine- Heme Onc
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| Lab |
Makowski Lab CRB 322
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| Street address |
19 South Manassas St
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| City |
Memphis |
| State/province |
Tennessee |
| ZIP/Postal code |
38163 |
| Country |
USA |
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| Platform ID |
GPL24247 |
| Series (2) |
| GSE174760 |
Bariatric surgery reduces obesity associated breast cancer and enhances response to immunotherapy [Mammary Fat Pad RNA-seq] |
| GSE174762 |
Bariatric surgery reduces obesity associated breast cancer and enhances response to immunotherapy |
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| Relations |
| BioSample |
SAMN19287029 |
| SRA |
SRX10948994 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5326659_A5738quant.sf.gz |
1.7 Mb |
(ftp)(http) |
SF |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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