title: GSM9209736: P14_Org_Apex_Bladder_R2; Canis lupus familiaris; RNA-Seq
library_construction_protocol: Organoids were preserved for bulk RNA-sequencing by removing them from Matrigel using Cell Recovery Solution, as previously described (Nicholson et al., 2025). The cell pellet was resuspended in 100 µL PBS, moved to a cryovial containing 900 µL RNAlater, and stored at -80°C. For RNA extractions, organoid samples were thawed and transferred to 15 mL tubes, washed with 2 mL of PBS, and centrifuged at 1,200 x g (4°C) for 5 minutes. Then the supernatant was removed and the cell pellet was resuspended in 1 mL of Trizol (Invitrogen; 15596026). Next, each sample was briefly vortexed. Tissues that were either flash frozen or stored in RNALater (if stored in RNALater a wash with PBS was done) were transferred from cryovials to microcentrifuge tubes, 800 µL of Trizol were added to each tube, and tissues were homogenized using a pestle. Both samples (tissue and organoids) were left at room temperature for five minutes prior to centrifuging at 12,000 x g (4°C) for 10 minutes. Supernatant was then transferred to a microcentrifuge tube, and 160 µL or 200 µL of chloroform (Alfa Aesar; J67241) were added for tissues and organoids, respectively. The samples were then vigorously mixed by shaking for 20 seconds and after sitting at room temperature for 2-3 minutes, the samples were spun at 10,000 x g for 18 minutes (4°C). The aqueous top layer was collected and moved to a sterile RNase-free tube before adding an equal amount of 100% RNA-free ethanol (Fisher; BP2818-500). Up to 700 µL were loaded in a Qiagen RNeasy column and collection tube (RNeasy Mini kit; Qiagen; 74104). Samples were then centrifuged at 8,000 x g for 30 seconds, the collection tube waste was discarded, and DNase treatment was performed according to Qiagen's protocol (Qiagen; 79254). In a new collection tube, 500 µL buffer RPE was added to the column, and the samples were again spun. After discarding the flow-through, this step was repeated, with samples now spun for 2 minutes at 8,000 x g. The flow-through was discarded again, and the samples were centrifuged at the same settings for 1 minute. Then, 50 µL of RNase-free water (Sigma; W4502-50ML) was added to the column and incubated for 2 minutes. Samples were centrifuged twice at 8,000 x g for 1 minute. RNA concentration was determined with a Nanodrop ND-1000 Spectrophotometer (Thermo Fisher Scientific), and samples were kept at -80°C until shipped. RNA extraction, library preparation, and sequencing were conducted at Azenta Life Sciences (South Plainfield, NJ, USA) as follows: RNA samples were quantified using Qubit 2.0 Fluorometer (ThermoFisher Scientific, Waltham, MA, USA) and RNA integrity was checked with 4200 TapeStation (Agilent Technologies, Palo Alto, CA, USA). Strand-specific RNA sequencing library was prepared by using NEBNext Ultra II Directional RNA Library Prep Kit for Illumina following manufacturer's instructions (NEB, Ipswich, MA, USA). Briefly, the enriched RNAs were fragmented for 8 minutes at 94 °C. First strand and second strand cDNA were subsequently synthesized. The second strand of cDNA was marked by incorporating dUTP during the synthesis. cDNA fragments were adenylated at 3'ends, and indexed adapter was ligated to cDNA fragments. Limited cycle PCR was used for library enrichment. The incorporated dUTP in second strand cDNA quenched the amplification of second strand, which helped to preserve the strand specificity. The sequencing library was validated on the Agilent TapeStation (Agilent Technologies, Palo Alto, CA, USA) and quantified by using Qubit 2.0 Fluorometer (ThermoFisher Scientific, Waltham, MA, USA) as well as by quantitative PCR (KAPA Biosystems, Wilmington, MA, USA). The sequencing libraries were multiplexed and clustered onto a flowcell on the Illumina NovaSeq instrument according to manufacturer's instructions. The samples were sequenced using a 2x150bp Paired End (PE) configuration. Image analysis and base calling were conducted by the NovaSeq Control Software (NCS). Raw sequence data (.bcl files) generated from Illumina NovaSeq was converted into fastq files and de-multiplexed using Illumina bcl2fastq 2.20 software. One mis-match was allowed for index sequence identification.