GSM3898664: J1 AQP5+; Homo sapiens; RNA-Seq
After at least three washes in HBSS, the pylorus was chopped finely and digested in incubation buffer supplemented with 1mg/mL Collagenase (Gibco) and 2mg/mL Bovine Serum Albumin (Sigma) for 30min at 37oC with intermittent mixing. Glands were isolated by repeated pipetting of finely chopped pylorus tissue in cold chelation buffer. Chelation buffer containing isolated glands was filtered through 100μm filter mesh, and centrifuged at 720g at 4oC for 3 min. The pellet was resuspended in TrypLE (Life Technologies) with DNaseI (0.8U/μL)(Sigma) and incubated at 37oC for 10 min with intermittent trituration for digestion into single cells. Digestion was quenched by dilution with cold HBSS buffer. The suspension was centrifuged at 720g at 4oC for 3 min. For anti-Aqp5 antibody stain, the pellet was resuspended in HBSS with 2% fetal bovine serum (FBS, Hyclone) with anti-Aqp5-AF647 (Abcam, ab215225) at 1:500 dilution and incubated on ice at 30min in the dark. The pellet was subsequently washed twice with cold HBSS and spun at 800g for 3min at 4oC. The pellet was resuspended in HBSS with 2% fetal bovine serum (FBS, Hyclone). Before sorting, 1 μg/ml propidium iodide (Life Technologies) was added to the cell suspensions, filtered through a 40μm strainer, and sorted on BD Influx Cell Sorter (BD Biosciences). Cells were collected in RLT Plus buffer (Qiagen) for subsequent RNA extraction with RNeasy Micro Kit according to manufacturer's instructions. Library was constructed with 10ng of input RNA using SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian according to manufacturer's instructions.
GEO Accession
GSM3898664