title: GSM8979597: CARLIN allele, injection at E7.5 and E8.5, collection at P4, otocyst cells, scRNA sequencing; Mus musculus; RNA-Seq
library_construction_protocol: Post natal mice were harvested in cold, sterile 1X CMF (Calcium Magnesium Free) PBS for each tissue extraction process. Following dissection of the inner ear, the tissue was dissociated via a 35-40 minute incubation period in 0.22 µm papain/ DNase solution at 37 deg C. Tissue was homogenized following dissociation by adding a 1:1 ratio of cold 1X CMF PBS + 4% FBS solution and pipetting up and down 150x per sample. Samples were centrifuged at 470 RCF for 5 min (at 4 deg), then supernatant was removed and replaced with fresh 1X CMF PBS + 4% FBS solution, re-suspending the pellet by pipetting. This process was repeated for more viscous samples. Cells then underwent a incubation step with a Apc-conjugated EpCAM antibody for 20 minutes at 4 deg, on a nutator. Finally, samples were filtered through 40 µm pluriSelect mini strainers into sterile 5 ml conical tubes. Purified APC+ cells from FACS were collected in 150 µl fresh 1X CMF PBS + 4% FBS solution per sample. All libraries were prepared according to the 10X Genomics protocol (Chromium™ Next GEM Single Cell 3' GEM, Library & Gel Bead Kit v3.1). In summary, Gel Beads-in-emulsion (GEMS) were produced via mixing Single Cell 3' v3.1 Gel Beads with the cell solution master mix for each sample and Partitioning Oil, then loading these into the Chromium Chip G. The gel beads were lysed and combined with primers containing a Illumina TruSeq R1 primer, a 12 nt unique molecular identifier (UMI), 16 nt 10X Barcode, and 30 nt poly(dT) sequence for each GEM, as well as a reverse transcription Master Mix of reagents. Following production of pooled, barcoded full-length cDNA from the poly-adenylated mRNA of each cell, the cDNA was purified from this mixture using silane magnetic beads, then amplified by PCR. Appropriately sized fragments were selected for the library, and paired end P5 and P7 primers, the TruSeq R2 primer sequence, and a sample index were added in the later stages of library construction. The library and cDNA were then used to generate the single-cell and bulk RNAseq samples respectively.