tissue type: primary breast tumor tumor stage: 4T1
Treatment protocol
not applicable
Growth protocol
67NR, 168FARN, 4T07, and 4T1 cells were cultured in medium supplemented with 10% FBS (Fetal Bovine Serum) in a CO2 humidified chamber at 37 °C. Three-month-old female BALB/c mice from Janvier Laboratory were used for cell line injection. 67NR, 168FARN, 4T07, and 4T1 cells (5 × 105) were harvested, rinsed in FBS-free medium, and injected into the fourth mammary fat pad in 100 µl of PBS. 8 mice were injected with 67NR cells, 11 mice were injected with 168FARN cells, 15 mice were injected with 4T07 cells, and 13 were injected mice with 4T1 cells. Primary tumors were excised once the average primary tumor size in each group reached 1- to 2-cm in size (measured at the largest dimension).
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated using Trizol total RNA isolation reagent (Invitrogen) according to the manufacturer's protocol.
Label
biotin
Label protocol
Samples were enzymatically fragmented and biotinylated using the WT Terminal Labeling Kit (Affymetrix)
Hybridization protocol
Samples were hybridized using Affymetrix hybridization kit materials • Heat cocktails at 99° for 5 minutes, then 42° for 5 minutes centrifuge at max speed for 1 minute (N.B. this deviates from Affy SOP). • Transfer 200μl of hyb solution to each array, then tape holes and parafilm. • Hybridize 16 hours at 45° at 60rpm• Fluidics washing is FS450_0001
Scan protocol
Affymetrix Gene ChIP Scanner 3000 7G
Description
4T1_rep3
Data processing
Data were processed using EASANA from GenoSplice technology. GC background correction were applied to probe intensities (core), exon level expression were summarized from the corrected intensities and then quantile normalized. Probe group file used in analysys: MoEx-1_0-st-v1.r2.pgf
