GSM8888760: B816 Lung Organoids; Canis lupus familiaris; RNA-Seq
Samples were expanded after thawing in CMGF + R/G and directly harvest as opposed to removing the R/G. Media was removed from wells and 500 µL of Cell Recovery Solution was added to each well, vigorously pipette mixed, and transferred to a 15 mL tube on ice for 20 minutes. The tube was then spun at 100 x g for 5 min at 4 °C and the supernatant was removed down to 500 µL. Then 1 mL of TrypLE Express was added to the sample, mixed, and the tube incubated in a 37°C heat bath for 8 minutes and shaken halfway through to mix the sample. After the incubation, the sample was pipette mixed to assist in dissociation of the organoids. Next, 7 mL of cold DMEM was used to stop dissociation. The sample was then strained through a pre-wet 40 µm filter and spun again. The sample was resuspended in 1 mL of DMEM and counted on an Invitrogen Countess 3 Automated Cell Counter. The sample was again spun, supernatant was removed, the pellet was resuspended in Cryostor CS10 and placed in a cryovial. The vial was immediately placed in a Mr. Frosty, incubated in a 4 °C fridge for 10 minutes, and transferred to a -80 °C for at least 24 hours. The samples were then either placed in liquid nitrogen for storage or shipped on dry ice. Single-cell RNA libraries were generated using the Chromium Single Cell 3' kit (10X Genomics, CA, USA). Loading onto the Chromium Controller was performed to target capture of ~6,000 GEMs per sample for downstream analysis and processed through the Chromium Controller following the standard manufacturer's specifications. The sequencing libraries were evaluated for quality on the Agilent TapeStation (Agilent Technologies, Palo Alto, CA, USA), and quantified using Qubit 2.0 Fluorometer (Invitrogen, Carlsbad, CA). Libraries were quantified using qPCR (Applied Biosystems, Carlsbad, CA, USA) prior to loading onto an Illumina NovaSeq XPlus instrument. The samples were sequenced at a configuration compatible with the recommended guidelines as outlined by 10X Genomics. Raw sequence data (.bcl files) were converted into fastq files and de-multiplexed using the 10X Genomics' cellranger mkfastq command.