||Surgical samples were collected from consenting breast cancer patients at Tan Tock Seng Hospital (Singapore) according to human subject research protocols approved by the Ethics Committee. Samples were washed with cold PBS with antibiotics three times, chopped with a sterile blade, and incubated by using MACS Tissue Dissociation Kits (Miltenyi Biotec) according to protocol. The resulting cell suspension were passed through 70 μm (BD Falcon, San Jose, CA, USA) and centrifuged at 800 rpm for 5 min at 4°C. cell were seeded in Single-cell suspensions (10,000 cell/well) were plated in 6-well ultra-low attachment plates (Corning) in DMEM/F12 medium containing 1X B27 (Invitrogen, cat. 12587-010), 50 ng/ml EGF, and 20 ng/ml βFGF for up to 1 months.
||Snap frozen human breast cancer tissues were disrupted using TissueLyser II (Qiagen). Total RNA including small RNAs from both human breast cancer tissue as well as the cell lines were isolated using miRNeasy Mini Kit (Qiagen, cat. 217004) according manual. In brief, c.a. 50 ng of total RNAs or 200 tumor cell were first lysed in reverse transcription buffer and the reaction is initiated with oligodT containing primer. Complete first strand synthesis is followed by template switching and the incorporation of SMARTer oligonucleotide. Full-length cDNAs are amplified using PCR to obtain DNA. Fragmentation and adapter introduction was performed using acoustic shearing to approximately 200 to 500 bp length and NEBNext® DNA Library Prep kit incorporating multiplex index primers. A pooled multiplexed library consisting equal amount of 6 individual libraries were sequenced on The HiSeq 2500 System by core facility.
|Library Construction Protocol:
|| RNA-seq libraries were generated by using the cDNA amplification kit SMARTer® Ultra™ Low RNA Kit (Cat. No. 634935, Clontech Laboratories, Inc. Mountain View, USA) for small amount of RNA or less than 200 cell according to manufacturer's manual followed by DNA library construction using the NEBNext® DNA Library Prep Master Mix Set for Illumina® kit (Cat. No. E6040S, New England Biolabs).