GSM9553558: Colorectal Cancer_Patient-Derived Organoid_Patient04; Homo sapiens; RNA-Seq
The organoid culture medium was removed, and 500 mL of organoid harvesting solution was added. The organoids were incubated on ice for 15 min, collected, and centrifuged at 400 ×g for 5 min at 4 °C. After discarding the supernatant, 200 mL of organoid digestion solution was added, followed by incubation at 37 °C for 5 min. Digestion progress was monitored to ensure dissociation into single cells; if necessary, the digestion time was extended. Once complete dissociation was achieved, digestion was terminated by the addition of 1 mL of advanced DMEM/F12  supplemented with 5 % FBS. The mixture was centrifuged at 400 ×g for 5 min at 4 °C. The pellet was resuspended in 5 mL of advanced DMEM/F12 and filtered through a 30 mm cell strainer. After centrifugation, the cells were washed twice with calcium- and magnesium-free DPBS. The resulting single-cell suspension was used for subsequent single-cell RNA sequencing. Within the Chromium instrument, barcoded Single Cell 3' Gel Beads are mixed with cells or nuclei, enzymes, and partitioning oil to form tens of thousands of single cell emulsion droplets called “GEMs” (Gel Bead-in-emulsion). Immediately following GEM generation, the Gel Bead is dissolved and any co-partitioned cell is lysed. Oligonucleotides containing (i) an Illumina Read 1 (read 1 sequencing primer), (ii) a 16 nt 10x Barcode, (iii) a 12 nt unique molecular identifier (UMI), and (iv) 30 nt poly(dT) sequence are released and mixed with the cell lysate and a Master Mix containing reverse transcription (RT) reagents. Incubation of the GEMs produces 10x Barcoded, fulllength cDNA from poly-adenylated mRNA. Then, the first-strand cDNA of 10X Barcoded was purified by silane magnetic beads, and sufficient mass was obtained by PCR amplification. The PCR products were clean up by SPRI beads, and double-stranded cDNA was obtained. Then quantify and qualify the dsDNA by Qubit3.0 Fluorometer (Invitrogen, Carlsbad, CA, USA) and Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA), so as to confirm the final PCR cycle number for library construction. Library Construction : The 1/4 of the dsDNA was then treated with Fragmentation Mix to fragment cDNA, End Prep Enzyme Mix to repair both ends and add a dA-tailing in one reaction, followed by double sided size selection to recover ~300 bp of the cDNA. Each sample was then added adaptors to both ends by a T-A ligation. The purified Ligation was amplified by PCR using P5 and P7 primers, with both primers carrying sequences which can anneal with flow cell to perform Illumina bridge PCR. And P7 primers are a mixture of four primers, each carrying a eight-base index allowing for multiplexing. The PCR products were cleaned up using beads, validated using an Qsep100 (Bioptic, Taiwan, China), and quantified by Qubit3.0 Fluorometer (Invitrogen, Carlsbad, CA, USA).