Reduction mammoplasty tissue was digested with Collagenase Type A (1 mg/mL, Roche) in DMEM/F12, 15 mM HEPES (1x penicillin/streptomycin, insulin (10 μg/ml), hydrocortisone (0.5 μg/ml)) for 14-16h. Retrieved organoids were dissociated with HyQtase (HyClone, Thermo Scientific) for 10 min at 37˚C and subsequent pipetting. To obtain single cells, cells were filtered twice through 40-μm cell strainers (Falcon). 10^6 cells were blocked for 10 min at 4°C with antibodies against human CD16 (FcRIII, Clone 3G8, 1:50) and CD32 (FcRII, Clone FUN-2, 1:100), washed and labeled in 100 μl medium for 20 min at 4°C with antibodies against human FITC-CD49f (Clone GoH3, 1:25), PerCP/Cy5.5-CD326 (EpCAM, Clone 9C4, 1:25), APC-CD10 (Clone HI10a, 1:20), PE-CD31 (Clone WM59, 1:33), PE-CD45 (Clone HI30, 1:33) and PE-CD235ab (Clone HIR2, 1:33). DAPI (0.2%, Invitrogen) was added (1:250) 2 min before cell sorting. Single cells were gated based on their forward and side scatter profiles. Dead cells (DAPI bright) and Lin+ cells (CD31+, CD45+ and CD235+) were gated out. 300 cells of a luminal epithelial (EpCAM+, CD49f+/-), basal epithelial (EpCAM-, CD49f+), CD10 negative and CD10 positive stromal cell population were directly sorted in 100ul TRIZOL in cooled tubes. The samples were immediately frozen on dry ice and stored at -70°C prior to RNA extraction.
Extracted molecule
total RNA
Extraction protocol
RNA was extracted from 300 sorted cells with the PicoPure RNA isolation kit (Arcturus)
Label
biotin
Label protocol
Reverse transcription was performed using 4 μM T7- (dT)24/T7- (dN)6 primer mix (Affymetrix) and 150 units of Superscript II reverse transcriptase (Invitrogen). The synthesis of second-strand cDNA was performed by mixing 4 mM dNTPs, 6 units DNA polymerase I, and 0.4 units RNase H in a 20-µL reaction volume. cRNA was produced by in vitro transcription with a T7 RNA polymerase at 37°C for 14 h using the MEGAscript T7 kit (Ambion) as per the manufacturer's instructions. For the second cycle, the first-strand cDNA was synthesized using 0.2 μg random primers from 9 μL of purified cRNA. The second-strand cDNA was produced using 10 µM T7- (dN)6 primer and 40 units DNA polymerase at 16°C for 2 h, after which 10 units T4 DNA polymerase (Invitrogen) were added and the incubation continued for another 10 min. The cDNA was in vitro transcribed with T7 RNA polymerase at 37°C for 16 h. The single-strand cDNA was synthesized using 10 µg purified cRNA in the presence of 4 µg random primers, 0.2 M DTT, 12 mM dNTP + dUTP and 750 units Superscript II (Roche Diagnostics) in a total volume of 20 µL. The cRNA was hydrolyzed with 2 units RNase H at 37°C for 40 min. The sense cDNA was purified and eluted in 28 µL elution buffer. Amplified products were purified using the GeneChip cDNA Sample Cleanup Module (Affymetrix) with a 6,000 g centrifugation during the first two steps. To improve recovery from the columns, water or elution buffer were spun into the matrix at 50 g and left to stand for 4 min before a 16,000 g centrifugation. The quantity and purity of the cRNA and cDNA produced during the first and second rounds were evaluated using a NanoDrop ND-1000 spectrophotometer (Nanodrop Technologies). The cDNA was then fragmented by uracil DNA glycosylase and apurinic/apyrimidic endonuclease 1 and biotin-labeled with terminal deoxynucleotidyl transferase using the GeneChip WT Terminal labeling kit (Affymetrix).
Hybridization protocol
Hybridization was carried out with 5 μg of biotinylated target, which was incubated with a GeneChip human Gene 1.0 ST Array (Affymetrix) at 45°C for 16 h. Following hybridization, non-specifically bound nucleotides were removed by washing and specifically bound target detected using a GeneChip Hybridization, Wash and Stain kit and a GeneChip Fluidics Station 450 (Affymetrix).
Scan protocol
The arrays were scanned using a GeneChip Scanner 3000 7G (Affymetrix) and CEL files acquired using GeneChip Command Console Software (Affymetrix).
Data processing
Arrays were normalized and probeset-level expression values calculated with R/Bioconductor's (v2.14) 'affy' package using the rma() function (www.bioconductor.org). Differential gene expression was determined using linear modeling implemented in the R/Bioconductor package ‘limma’. For general analysis, a cutoff of linear fold change >2, and pvalue <0.005 were used.
