NextSeq 500 paired end sequencing; scRNAseq of two medulloblastoma cell lines (ONS-76 and HDMB-03) grown in 3D hydrogel culture
scRNAseq of two medulloblastoma cell lines (ONS-76 and HDMB-03) grown in 3D hydrogel culture
Four 3-week hydrogel cultures of ONS-76 and HD-MB03 cells (pools of three gels per cell lines) were transferred into gentleMACS C tubes and processed using the Tumour Dissociation kit and the GentleMACS Dissociation programme (all Miltenyi Biotec) according to the manufacturer's instructions. Dead cells and cell debris were removed from the resulting suspension of single cells using MS columns and the Dead Cell Removal Kit (both Miltenyi Biotec) according to the manufacturer's instructions. Library preparation and sequencing were performed by the Deep Sequencing facility of the University of Nottingham. Briefly, single cell 3' whole transcriptome sequencing libraries were prepared from dissociated cell suspensions using the Chromium Single Cell 3' Library and Gel Bead Kit v2, the Chromium Single Cell A Chip Kit and the Chromium Multiplex Kit, 96 rxns (10X Genomics, Pleasanton, California, USA; PN-120267, PN-1000009 and PN-120262). Cell counts and viability estimates were obtained using the LUNA-II Automated Cell Counter (Logos Biosystems), Trypan Blue Stain, 0.4 % and Luna Cell Counting Slides (Logos Biosystems; T13001 and L12001). Live cell counts were used to calculate cell input, rather than total cell count, as visual inspection of cell field on the LUNA II and the gating histogram, showed that > 95 % of cells looked viable and that the cell counter appeared to be counting some extracellular debris as non-viable cells. Five thousand input cells were targeted per sample, with the aim of generating sequencing libraries from ~ 2,500 single cells. All steps, including GEM Generation and Barcoding, Post GEM-RT Cleanup and cDNA Amplification and Library Construction were performed according to the Chromium Single Cell 3' Reagents Kits v2 User Guide, Rev D. Variable steps of this protocol included using 12 cycles of cDNA amplification and 10 cycles of library amplification. Amplified cDNA was quantified using the Qubit Fluorometer and the Qubit dsDNA HS Assay Kit (ThermoFisher Scientific; Q32854) and fragment length profiles were assessed using the Agilent 4200 TapeStation and Agilent High Sensitivity D5000 ScreenTape Assay (Agilent; 5067-5592 and 5067-5593). Completed sequencing libraries were quantified using the Qubit Fluorometer and the Qubit dsDNA HS Assay Kit and fragment length distributions assessed using the Agilent 4200 TapeStation and the High Sensitivity D1000 ScreenTape Assay (Agilent; 5067-5584, 5067-5585).