GSM8415307: NP_23_B; Mus musculus; RNA-Seq
L2-5 and R2-5 mammary glands were harvested, minced, and chemically digested overnight in Advanced DMEM F/12 with 1% PSA, gentle collagenase/hyaluronidase, and DNAse I at 37C, 5% CO2, and added humidity. Partially digested glands were then mechanically digested by pipetting with a serological pipette until no tissue pieces were visible. Digested glands were washed with staining buffer (Hank's Balanced Salt Solution, 2% Bovine Calf Serum, 1% PSA) and centrifuged (1500RPM) at 4C for 5 minutes. Red blood cells were lysed with 5mL of ACK Lysis buffer for 5 minutes, and cells were washed with 15mL of staining buffer. Cells were treated with 0.25% Trypsin with EDTA and gently pipetted continuously for 2-3 minutes to digest the basement membrane. Cells were then treated with DNAse I and Dispase and pipetted continuously for 2-3 minutes to prevent clumping. The single-cell suspension was then filtered through a 40um mesh strainer and pelleted via centrifugation (1500RPM, 4C, 5 minutes). RNA was isolated according to manufacturers instructions (Qiagen RNEasy Plus Micro Kit No. 74034). Messenger RNA was purified from total RNA using poly-T oligo-attached magnetic beads. After fragmentation, the first strand cDNA was synthesized using random hexamer primers, followed by the second strand cDNA synthesis using either dUTP for directional library or dTTP for non-directional library80. For the non-directional library, it was ready after end repair, A-tailing, adapter ligation, size selection, amplification, and purification. For the directional library, it was ready after end repair, A-tailing, adapter ligation, size selection, USER enzyme digestion, amplification, and purification. The library was checked with Qubit and real-time PCR for quantification and bioanalyzer for size distribution detection. Quantified libraries will be pooled and sequenced on Illumina platforms, according to effective library concentration and data amount.