GSM9962060: Pancreatic tumor, vehicle only control, fish 3; Danio rerio; RNA-Seq
Fish are placed in ice water to euthanize following Dartmouth IACUC procedures. An en bloc dissection method was used to remove all organs intact. Briefly, the head was removed with scissors and intestines removed with forceps. Tumors were imaged en bloc and removed intact by peeling away surrounding organs/tissue, then sectioned in half and thoroughly rinsed in cold PBS. Half of the tumors were placed directly in Dissociation Mix w/ RNase inhibitor; the other half of the tumor was placed in 4% PFA for histology (rocking at 4˚C overnight). Dissociation Mix was Liberase TM Research Grade (Sigma Cat #5401119001) [final concentration 0.15mg/ml] prepared in 1X HBSS (w/ calcium & magnesium) with DNAse I (Zymo Cat #E1011) [final concentration 0.002U/uL] and RNase inhibitor (Roche Cat #3335402001) [final concentration 1U/ul], heated to 28˚C before use. Tumors in Dissociation Mix were placed in a thermomixer set at 28˚C 800rpm for 20–30-minute intervals. Tissue was mechanically dissociated with a pipette between intervals. Dissociation was stopped with FBS and washed 3X in Wash Buffer w/ RNase Inhibitor (1X PBS (calcium and magnesium-free) containing 0.04% weight/volume BSA (Cat # B6917-100MG) and a final RNase Inhibitor concentration of [0.5U/ul]). The samples were filtered through a 40um nylon mesh and dead cells were removed using the Miltenyi Dead Cell Removal Kit (Cat #130-090-101) and Miltenyi MS Columns (Cat #130-042-201). Cells were counted with trypan blue and resuspended in Wash Buffer w/ RNase Inhibitor. Samples were placed on ice prior to sequencing. Following the tumor dissociation, samples were processed in the Dartmouth Genomics Shared Resource Core using a Chromium X Controller and Universal 3' gene expression kit for cell capture and library preparation (10 X Genomics). Libraries were sequenced on a NextSeq2000 sequencer (Illumina) and reads aligned to the Danio rerio GRCz11 reference genome with Cell Ranger (version 8.0.0). Samples averaged ~10K cells captured with ~30K reads per cell.