title: GSM9564520: Mullerian duct cells E6.5, male and female, CellPlex; Gallus gallus; RNA-Seq
library_construction_protocol: The Müllerian duct tissues were dissociated by adding 300µL of 0.25% Trypsin-EDTA to each tube, and incubating at 37°C for 18 minutes, shaking at 750RPM. The digestion was stopped with 75µL of neat Fetal Calf Serum (FCS). The dissociated cells were filtered by loading onto 35µm Cell Strainer FACS tubes. The strainer was washed twice by adding 200µL 10% FCS in PBS, and centrifuging at 600G, room temperature, for 1 minute. The cells were pelleted by spinning at 600G, room temperature for 3 minutes. The 10% FCS in PBS was removed, and the cells were resuspended in 1mL 0.04% BSA in PBS. The cells were spun at 400G, room temperature, for 5 minutes. The following CMOs (10X Genomics) were used to label three technical replicates for each sample: E6.5 Mixed Sex (CMO 304-306), E8.5 Female (CMO 307-309), E8.5 Male (CMO 310-312), E11.0 Female left duct (CMO 301-303), E11.0 Female right duct (CMO 304-306 for whole ducts, CMO 307 for duct tips). The CMO labelling protocol was carried out in accordance with the manufacturer's instructions (10X Genomics document CG000391 Revision B). Each technical replicate was resuspended in 100µL CMO and incubated for 5 mins at room temperature. Following CMO labelling, the dissociated cells were washed three times in 1% BSA in PBS, on ice. Each technical replicate was diluted to 1000 cells/µL and determined to be of >95% viability using trypan blue staining and the Countess 3 Cell Counter. Each technical replicate was pooled in equal proportions into a single sample. For each sample, 16,500 cells at 1000 cells/µL were loaded to obtain a targeted recovery of 10,000 cells post-sequencing. Single-cell analysis was carried out using the 10X Genomics Chromium Controller and Chromium Single Cell 3′ Library & Gel Bead Kit V3.1, as per the manufacturer's instructions (10X Genomics document CG000388 Revision D). Samples were subjected to 11 cycles of PCR for cDNA amplification and 13/14 cycles for library amplification. Completed libraries were pooled in an equimolar ratio along with a 5% PhilX Control Library V3, per the manufacturer's instructions (Illumina document 15048776 V11). The prepared libraries were sequenced using an Illumina Novaseq at Azenta Life Sciences, using SBS chemistry, in paired-end (PE)-150 configuration, targeting 20,000 read pairs per cell for gene expression libraries and 5000 read pairs per cell for Cell Multiplexing libraries.