GSM8513207: Med13L WT_cortex_E13.5, scRNA; Mus musculus; RNA-Seq
For bulk RNA sequencing, total mRNA from mouse cortical tissues was isolated using the RNAiso Plus kit (Takara, #9109) on ice. For scRNA sequencing, cortex from 5 embryos of the same genotype from 3 litters were pooled into 5 ml tissue storage solution (Miltenyi Biotec, Inc, MD, USA) on ice and carefully cut into small pieces of 1-3 mm. Tissue samples were subjected to enzymatic digestion with 0.25% Trypsin-EDTA (Thermo Fisher Scientific) at 37°C in an atmosphere of 5% CO2 for up to 30 min. Cell suspensions were centrifuged at 300 × g for 5 min at 4℃. The pellets were further digested in basal media supplemented with 0.2% type II collagenase (Sigma-Aldrich) at 37°C for up to 4 h. Isolated cells were filtered through 70 mm nylon filters (BD), washed twice with sterile PBS. Samples with a cell density of 700-1200 cells/ml and a viability higher than 85% were proceeded for scRNA-seq. For bulk RNA sequencing, RNA-seq libraries were constructed using the VAHTS Universal V10 RNA-seq Library Prep Kit (Premixed Version, NR616). For scRNA sequencing, Droplet-based scRNA-seq was performed using the 10x Genomics Chromium Single Cell 3′ Reagents Kit (v3.1). The single cell samples were passed through a 40 μm cell strainer and counted using a hemocytometer with trypan blue. Subsequently, the single cell suspension, Gel Beads and oils were added to the 10x Genomics single-cell A chip. After droplet generation, samples were transferred into PCR tubes and reverse transcription was performed using a T100 Thermal Cycler (Bio-Rad). After reverse transcription, cDNA was recovered using a recovery agent, provided by 10x Genomics, followed by silane DynaBead clean-up as outlined in the user guide. Before clean-up using SPRIselect beads, cDNA was amplified for 10 cycles. Libraries were submitted to 150 bp paired-end sequencing on an Illumina Novaseq 6000 platform, yielding 150 G of sequencing data per sample.