Transcriptome analysis of MCF-7 breast cancer cell population to reveal the transcriptional diversity at the single cell level
Transcriptome analysis of MCF-7 breast cancer cell population to reveal the transcriptional diversity at the single cell level
MCF-7 breast cancer cells were purchased from Bioresource Collection and ResearchCenter (BCRC) of Taiwan (BCRC 60436, Lot-01337) and cultured in DMEM medium (GIBCO11955-065) supplemented with fetal bovine serum (GIBCO 26140-079, 10% v/v) and insulin(Sigma I0516, 5 g/ml), in the presence of penicillin (100 unit/ml) and streptomycin (GIBCO15140-122, 100 mg/ml). Cell cultures were maintained in T150 flasks (Cloning) inside a37C humidified incubator with 5% CO2. Culture media were changed every 3 days. Single cells were picked up by mouth pipetting under a microscope. Each single cell was transferred into a 0.2 ml thin-walled PCR tube which already contains cell lysis buffer (ref. to sequencing library preparation protocol provided by Applied Biosystems (AB)). The initial volume of the cell-containing solution should be kept below 0.5 ul to minimize the dilution effect and each glass pipette should be used only once. Single cell library was constructed by following AB's protocol, but with following modifications: i) SuperScript II, instead of SuperScript III, was used as the reverse transcriptase during reverse transcription with UP1 primer; ii) accordingly, the incubation conditions in the same step was changed from 50C for 30 min to 42C for 50 min; and iii) duration time of the first PCR amplification was extended from 6 min to 8 min. PCR products were displayed by 1.5% agarose gel electrophoresis, and 0.5-3 kb cDNA molecules were isolated for sequencing library construction. Besides the 5000-cell library (fM7_tot50ng), a total of four SCT libraries (fM7_sC4, fM7_sC5, fM7_sC6, and fM7_sC8) wereconstructed and sequenced with SOLiD 3 sequencer for 35 ligation cycles.
Experimental Factor: INDIVIDUAL
sC6