GSM8833703: Chicken 7 Organoids; Gallus gallus; RNA-Seq
Organoids were harvested for single-nuclei RNA sequencing via collection from the Matrigel matrix using the cleaning protocol previously described. Organoids were resuspended in 500 µL DMEM/F12 and transferred to a cryovial. After centrifuging at 100 x g (4°C) for 5 minutes, the supernatant was removed, and the sample was flash-frozen in liquid nitrogen for 30 seconds. Samples were stored in a -80°C freezer and sent to Genewiz from Azenta Life Sciences (South Plainfield, NJ, USA) on dry ice for analysis. Upon arrival, tissue and organoid samples were stored in liquid nitrogen for further processing. Using the manufacturer's instructions, nuclei were extracted from both samples using Miltenyi Nuclei Extraction Buffer (Miltenyi Biotec, Auburn, CA, USA), in addition to gentle MACS Dissociation and C tubes. Once isolated, AO/PI dye was used on the Nexcelom Cellaca MX to count the nuclei, and a Chromium Single Cell 3' kit (10X Genomics, CA, USA) was used to create single nuclei RNA libraries. Following the manufacturer's guidelines, the nuclei were loaded and processed on the Chromium X, targeting approximately 6,000 gel bead-in-emulsions (GEMs) per sample. An Agilent TapeStation (Agilent Technologies, Palo Alto, CA, USA) evaluated quality of the sequencing libraries, and a Qubit 2.0 Fluorometer (Invitrogen, Carlsbad, CA, USA) quantified them. qPCR (Applied Biosystems, Carlsbad, CA, USA) was also used to quantify libraries before loading the samples on an Illumina HiSeq 4000 (or equivalent) instrument. Then, samples were sequenced using the 10X Genomics recommended guidelines. The raw sequence data (.bcl files) were converted to fastq files before being de-multiplexed with the Genomics' cellranger mkfastq command.
GEO
GSM8833703_r1