title: GSM9245094: IntestiCult™ Plus, Duodenum, 10X, rep1; Homo sapiens; RNA-Seq
library_construction_protocol: Intestinal organoids in matrigel domes were digested using gentle cell dissociation reagent for 10 minutes, followed by centrifugation at 300g for 5 minutes. The cell pellet is washed with DMEM-F12 + 0.5% BSA, and centrifuged at 300g for 5 minutes. After removal of the supernatant, the pellet is resuspended in 1mL of warmed (37°C) Trypsin-EDTA (0.05%), and incubated at 37°C for 5-10 minutes. The cell suspension is then mixed thoroughly until large fragments have dissociated into single cells. An equal volume of DMEM/F-12 + 1% BSA is added to the suspension to deactivate the trypsin. A pre-wet a 37um reversible strainer is used to remove any large fragments that remain. Cell concentration, viability and percent of clumps are determined using a Nucleocounter. Single cells are centrifuged at 200g for 5 mins (RT). The supernatant was removed, and 1mL of pre-warmed cell suspension buffer is added with a wide bore pipette and gentle mixing to resuspend the pellet. Cells are then centrifuged at 200g for 3 minutes (RT), followed by aspiration of the supernatant. 200-400uL of cold cell suspension buffer is added to the pellet and gently mixed 10-15 times to resuspend. The single cell concentration is determined using the laboratory's preferred method. A suspension of 2,500 live cells/uL is prepared in a cell suspension buffer to achieve an input volume of 4uL (10,000 live cells/uL) into a PIP reaction. 10X libraries: Library was prepared using Chromium Single Cell 3ʹ v1 protocol with GEM-X Universal Multiplex technology (10x Genomics). Sample quality control was performed using the Agilent 2100 Bioanalyzer or the Agilent 4200 TapeStation. Qualifying samples were then prepped following the standard protocol for the Illumina Stranded mRNA prep (Illumina). Sequencing was performed on the Illumina NextSeq 2000 with Paired End 59 bp × 59 bp reads. Illumina libraries: Library preparation was performed according to the manufacturer's instructions (Illumina Single Cell 3′ RNA Prep, T10, Illumina). Briefly, single cells were combined with Particle-templated Instant Partitions (PIPs) and barcoded beads to generate emulsified partitions. Following chemical lysis, mRNA released from each partition was reverse transcribed into barcoded cDNA. The cDNA was then amplified by whole transcriptome amplification, isolated from PIPs, and purified using magnetic beads. Quality control included Qubit quantification and fragment analysis. For library construction, cDNA underwent fragmentation, end repair and A-tailing, adapter ligation, and sample index PCR to add Illumina P5, P7, and index sequences. Final libraries were cleaned with magnetic beads and prepared for sequencing on Illumina platforms.
identifiers.namespace: GEO
identifiers.id: GSM9245094_r1