title: GSM7114132: wildtype mTECs, ALID09; Mus musculus; RNA-Seq
library_construction_protocol: For the transcriptional time course, cells were collected on day one, three, nine, and thirty-six at air/liquid interface. Collection involved washing mTECs with PBS three times both apically and basally, incubating in 0.05% Trypsin-EDTA in Cell Dissociation Buffer (Gibco, Cat #13151014) added both apically and basally at 37°C for 15 min, gentle trituration, and inactivating trypsin in mTEC Basic with 2% Nu-Serum (Corning, Cat #355100). Additional 0.05% Trypsin-EDTA in Cell Dissociation Buffer was added to the apical surface and incubated at 37°C for 5 min. Cells were washed and resuspended in 0.04% BSA in PBS at 1000 cells/L. Library was performed according to the manufacter's instructions (single cell 3' v3.1 protocol, 10x Genomics). Briefly, 10,000 (ALID01) or 25,000 (ALID03, ALID09, ALID36)  mTECs were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip to generate the gel bead-in-emulsion (GEM). The poly-A RNA from the cell lysate contained in every single GEM was retrotranscripted to cDNA, which contains an Ilumina R1 primer sequence, Unique Molecular Identifier (UMI) and the 10x Barcode. The pooled barcoded cDNA was then cleaned up with Silane DynaBeads, amplified by PCR and the apropiated sized fragments were selected with SPRIselect reagent for subsequent library construction. During the library construction Ilumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added.