title: GSM8958233: Tumor, LN2A, rep 3; Mus musculus; RNA-Seq
library_construction_protocol: Mice were sacrificed via cervical dislocation and then tumors were dissected from the lung and placed in cold DMEM + Y27632 (ROCK Inhibitor, Selleckchem #S1049) (1:1000) and DNAse I (Worthington Biochemical Corporation #LS002058) (1:50) until ready for dissociation. Tumor tissue was cut into small pieces then dissociated in a solution of HBSS, Y27632 (1:1000), DNase I (1:50) and Protease from Bacillus licheniformis (Sigma #P5380) (1:20). The tumor suspension was triturated through a cut pipette tip then placed on a rocker in a cold room. This process was repeated every five minutes until the tumor was visibly broken down. Cells were filtered through a 70 μM filter and washed with fully supplemented DMEM. Cells were pelleted and cryopreserved with FBS + Y27632 (1:500), DNase I (1:50) and DMSO (1:10). Dissociated tumor cell viability of each sample was checked by acridine orange and propidium iodide staining using the LUNA-FL Dual Fluorescence Cell Counter (Logos Biosystems) and concentrated to 800 live cells/μL in 0.04% BSA in PBS. The 10X Genomics Chromium Controller and the Chromium Single Cell 3' GEM, Library & Gel Bead Kit v3 (PN-100075) were used for processing cells according to the manufacturer's recommendations (Document#CG000183 Rev C). Library preparation was done by loading approximately 16,000 cells per sample onto the Chromium Chip B, targeting a 10,000-cell recovery per sample. Gel beads in emulsion (GEMs) were created by encapsulating single cells, reverse transcription reagents and gel beads coated with barcoded oligos in an oil droplet. Reverse transcription was used to create cDNA libraries tagged with a cell barcode and unique molecular index (UMI). cDNA libraries were purified using Dynabeads MyOne SILANE (Invitrogen) before 11 amplification cycles after GEMs were broken. Amplified cDNA libraries were purified with SPRIselect magnetic beads (Beckman Coulter) and quantified using an Agilent Bioanalyzer High Sensitivity DNA chip (Agilent Technologies). After these SPRIselect beads were used for fragmentation, end repair, A-tailing and double-sided size selection, Illumina-compatible adapters were ligated onto the size selected cDNA fragments. These were then purified and amplified with uniquely identifiable indexes. After purification, the finalized sequencing libraries were visualized using the Bioanalyzer High Sensitivity DNA chip, normalized to 20 nM, and pooled.
identifiers.namespace: GEO
identifiers.id: GSM8958233_r1