Instrument: Illumina HiSeq 4000
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: PAIRED
Construction protocol: Basal lingual epithelial cells were purified from E16 embryos. Tongues were removed from the mandible and cut to exclude the posterior circumvallate papillae. For RNA and RNA-Seq analysis of control, Krt14-Cre Ring1a-/- Ring1b flox/flox mice, E16 tongues were collected. Tissues were cut into small pieces and incubated with 1.26U/mL dispase (Invitrogen) and 0.3% type 1 collagenase (Worthington) for 45 minutes at 37ºC with 80 rpm shaking. Tissues were washed with 1xPBS, dissociated with 0.25% Trypsin with 2.21mM EDTA (Corning Cellgro; Manassas, VA, USA), and then washed twice with 1xPBS. Cells were stained with 1:200 EpCAM-APC antibodies (Biolegend; San Diego, CA, USA) for 30 min on ice and washed twice with 1x HBSS prior to cell sorting. For ChIP analysis, control newborn P0 mice were collected. All cell isolations were performed on a FACS Influx instrument (BD, Franklin Lakes, NJ, USA). FACS-purified cells were collected directly into RLT Plus buffer (QIAGEN), and total RNA was isolated with the RNeasy Plus Micro Kit (QIAGEN). Complimentary DNA was reverse-transcribed from total RNA using qScript cDNA SuperMix (Quanta Biosciences, Gaithersburg, MD, USA) according to the manufacturer's instructions. 15ng of RNA were reverse transcribed and amplified using the Ovation RNA-seq System V2 (Nugen). Libraries were constructed from 50ng of sonicated cDNA (Covaris) using the Ovation Ultra Low DR Multiplex system (Nugen). The concentration and quality of the libraries were determined using Qubit (Invitrogen) and Bioanalyzer (Agilent). Constructed RNA-seq libraries were sequenced at GENEWIZ on the Illumina HiSeq platform, obtaining 100-nucleotide single reads.