title: GSM4495753: Mixed samples (A,B,C) Day 3_scRNA-Seq; Mus musculus; RNA-Seq
library_construction_protocol: [media]  A: Cells were removed from collagen matrix by a 10 min incubation on a benchtop shaker at 180 r.p.m. (37 °C) with 2 mg ml−1 collagenase in PBS, B:For Matrigel matrix, gels were washed once with ice-cold PBS without Ca2+ and Mg2+ followed by a 30 min incubation on ice with 10 mM EDTA (Sigma, EDS) [dissociation into single cells]  A,B:After two washes in PBS, organoids were dissociated into single cells by incubating with 10X TryplE (Thermo Fischer, A1217701), 10 min on a benchtop shaker at 180 r.p.m. (37 °C). After centrifugation at 400g for 10 min, 2 U μl−1 DNase (Sigma, D4263) were added in DMEM-F12 and incubated for 5 min at room temperature. Cells were resuspended in PBS and filtered through a 40-μm filter to remove the undigested organoids;C:4T1 cells were trypsinized using trypsin-EDTA 0.25 % (Thermo Fisher, 25200056). Detached cells were resuspended in culture media and centrifuged at 400g, 5 min at room temperature. [barcoding protocol]  Cells were barcoded by condition with MultiSeq (McGinnis et al., 2019) and flow sorted for live cells using Propidium iodide (Thermo Fisher, P3566). Multi-seq library was prepared following 10X single cell controller protocol and Multi-Seq specific protocol. Library construction from the single cell suspensions were performed using the 10X Genomics Chromium Single Cell 3' Reagent Kit v2, following manufacturer's instructions.Briefly, Multi-Seq barcoded and pooled single cell suspension were counted using Countess II (Thermal Fisher) to determine cell concentration and viability. The cell volume to use was calculated based on the cell concentration and the targeted cell number. The cell suspension was brought to 46.6 µl with molecular biology water and then mixed with 33.4 µl premade single cell master mix containing RT reagent, template switch oligo, reducing reagent, and RT enzyme.  75 µl of the resulting cell/master mix solution were loaded to 10X single cell A Chip row 1. The gel beads were vortexed for 30 seconds and 40 µl were loaded to 10X single cell A Chip row 2.  280 µl partition oil were loaded to 10X single cell A Chip row 3. The unused lanes were filled on the Chip with 50% Glycerol. 10X provided gasket was attached to the chip, then the chip was loaded into 10X single cell controller and the cell partitioning program was run. After the program completes, 100 µl of GEM were removed from the recovery well and slowly dispensed into pre-chilled PCR plate. The plate was sealed and incubated at 53°C for 45 min, 80°C for 5 min and then chill to 4°C. The recovery reagent was then added to the GEM-RT reaction to break up the single cell emulsion and the cDNA was purified using Thermal fisher Dynabeads MyOne Saline magnetic beads and amplified by PCR using 10X provided reagents and Multi-Seq primer. The amplified cDNA was purified using Beckman Coulter SPRI magnetic beads and the endogenous transcript cDNA and Multi-Seq barcode cDNA were separated. The purified cDNA was quantitated using Thermal Fisher Qubit high sensitivity dsDNA assay and ran on Agilent Bioanalyzer High sensitivity DNA chip for quality control. The transcript cDNA library was prepared following standard Illumina library preparation using 10X provided reagents, i.e., fragmentation, end repair and dA tailing, adapter ligation, and PCR amplification. The Multi-Seq barcode cDNA library was prepared by PCR amplification using universal i5 primer and RPI index primer. After library qc and quantitation, the transcript library and the Muti-Seq barcode library were pooled in 95:5 ration and sequenced together on NovaSeq.
identifiers.namespace: GEO
identifiers.id: GSM4495753