GSM9082759: Tracheal cells, E14.5; Mus musculus; RNA-Seq
To prepare single-cell suspensions of mouse tracheal cells, tracheas were dissected from E11.5-E14.5 embryos. Dissected tracheas were incubated on a microtube shaker with mixture of 0.25% Trypsin-EDTA (Thermo Fisher Scientific) and 4 mg/mL collagenase type Ⅱ (Worthington) at 37 ˚C for 15 – 45 min with trituration by a P1000 pipette every 15 min. 1 mL PBS containing 10% FBS was added to quench the enzymes. The cell-suspensions were loaded onto a 40 µm cell strainer (Falcon) to purify the single cells. Single-cell suspensions were pelleted by centrifugation at 300 x g for 3 min in a swinging-bucket rotor. The supernatant was removed, and cells were resuspended in PBS with 0.5% bovine serum albumin (BSA). Single cell sequencing library construction were performed using the 10x Genomics Chromium Single Cell 3' Library & Gel Bead Kit following the manufacturer's instructions. Libraries were sequenced on a HiSeq X (Illumina).