GSM9421120: E14, 3D culture, mylk-shRNA, mCherry-negative, litter 3; Mus musculus; RNA-Seq
After 72 hours, kidney explant cultures were pooled per condition and digested using a solution composed of 1:1 (v/v) 0.25% trypsin-EDTA and 1% collagenase. Digests were carried out for 30 minutes in a humidified incubator set to 37oC and 5% CO2. Digests were quenched using FBS set to 10% volume and washed in FACS media (DPBS with 2% FBS and 1% P/S) prior to sorting. Cells within the top 25% of mCherry signal were collected as the positive fraction, and cells within the bottom 50% of mCherry signal were collected as the negative fraction. Cells were washed once with DPBS prior to total RNA isolation using a Rneasy mini plus kit (Qiagen). Libraries were prepared and sequenced using a commercial service (Plasmidsaurus). In brief, 100-300 ng total RNA per sample was subject to mRNA capture, reverse transcription, and second strand synthesis. This was followed by tagmentation and Illumina library indexing and amplification.