title: GSM8833692: Chicken 3 Organoids_rep2 [RO-38]; Gallus gallus; RNA-Seq
library_name: GSM8833692
library_construction_protocol: After at least 4 passages, organoids were preserved for bulk RNA-sequencing by following the cleaning protocol described in the development and maintenance of organoid cultures section. After removing 6 mL of DMEM/F12 supernatant, the cell pellet was resuspended in 100 µL PBS, moved to a cryovial containing 900 µL RNAlater (Invitrogen; AM7021), and stored at -80°C. For RNA extractions, the organoids 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. The supernatant was removed, the cell pellet was resuspended in 1 mL of Trizol (Invitrogen; 15596026), and each sample was briefly vortexed. Flash frozen tissues were transferred from cryovials to microcentrifuge tubes, 800 µL of Trizol were added to each tube, and tissues were pestled until homogenized. Both tissue and organoid samples were left at room temperature for five minutes before centrifuging at 12,000 x g (4°C) for 10 minutes. The supernatant was 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 vigorously mixed by shaking for 20 seconds. After sitting at room temperature for 2-3 minutes, 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. Up to 700 µL were loaded in a Qiagen RNeasy column and collection tube (RNeasy Mini kit; Qiagen; 74104). Samples were centrifuged at 8,000 x g for 30 seconds, the waste in the collection tube 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 spun at the same settings as before. After discarding the flow-through, this step was repeated, but samples were 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. 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. All samples were sent to Azenta Life Sciences (South Plainfield, NJ, USA) for preparation of the RNA library and bulk RNA sequencing. A Quibit 2.0 Fluorometer (Life Technologies, Carlsbad, CA, USA) was first used to quantify RNA, followed by assessment of the RNA integrity with an Agilent TapeStation 4200 instrument (Agilent Technologies, Palo Alto, CA, USA). A NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (NEB, Ipswich, MA, USA) was used to prepare the RNA sequencing libraries, using the manufacturer's instructions. Then the mRNA was enriched with Oligo(dT) beads and fragmented for 15 minutes (94°C). Both cDNA strands were synthesized, and at the 3' ends, fragments were end-repaired and adenylated. The cDNA fragments were ligated with universal adapters, and limited-cycle PCR was used for index addition and library enrichment. An Aligent TapeStation (Agilent Technologies, Palo Alto, CA, USA) was utilized to confirm the sequencing libraries, and both a Qubit 2.0 Fluorometer (Invitrogen, Carlsbad, CA) and quantitative PCR (KAPA Biosystems, Wilmington, MA, USA) were used for quantification. Sequencing libraries were multiplexed and clustered onto one flow cell. Using the manufacturer's protocol, an Illumina instrument was then used to sequence the samples with 2x150bp Paired End (PE) configuration. Control Software (HCS) was used for image analysis and base calling. Illumina HiSeq created raw sequence data (.bcl files), which were converted to fastq files and de-multiplexed with Illumina's bcl2fastq 2.17 software. For index sequence identification, one mismatch was allowed.
identifiers.namespace: GEO
identifiers.id: GSM8833692_r1