This protocol is for performing single cell RNA sequencing (scRNAseq) on ovarian tumor samples. We are using the Miltenyi MACS dissociator system for generating single cell suspensions and then sequencing using 10X genomics 3' chromium system followed by Illumina NovaSeq.
Hypothermosol. BioLife Solutions Cat # 101102
RPMI-1640 or DMEM
FBS
DMSO
RNALater
gentleMACS Dissociator
MACS C Tubes. Miltenyi
MACS SmartStrainer (70µm). Miltenyi
Tumor Dissociation Kit, human. MACS Cat # 130-095-929
Red Blood Cell Lysis Buffer (100 ml). Roche Cat # 11 814 389 001
Dissecting scissors and/or scalpel
Forceps
15 ml conical tube
Freezing tubes
Coordinate with BioNet prior to surgery to setup sample collection. Provide BioNet with 50 ml conicals containing DMEM and instruct BioNet to store conicals on ice.
Retrieve samples in 50 ml conicals from BioNet collection room in hospital
Take pictures of the tubes/samples and record the following:
Date and time of collection: ___________________________________
BioNet Sample ID: _____________________________________________
Gross weight of tissue (g): ____________________________________
Description of tissue: ______________________________________________________________________________
______________________________________________________________________________________________________
Perform all procedures in a BioSafety Cabinet and use aseptic techniques.
Place sample in 10 cm petri dish sitting on ice and rinse with 4ºC PBS. Use scissors or scalpel to remove any visibly necrotic tissue or clearly non-tumor tissue. Weigh remaining viable cancer tissue
Gross weight of viable cancer tissue (g): ________________________________________
Cut the sample into the following aliquots and store accordingly:
1-6 g for single cell sequencing. Note: 1 g is considered minimum desireable quantity, but we have processed samples as small as 0.1 g and were able to get scRNAseq data. Weigh the portion used for single cell. See below for single cell dissociation procedure.
Chunk for DNA. Place in 1.5 ml cryovial and put cryovial in liquid nitrogen. After sample is frozen store at -80ºC. Size of chunk will depend on how much tissue we received.
Chunk for Protein. Place in 1.5 ml cryovial and put cryovial in liquid nitrogen. After sample is frozen store at -80ºC. Size of chunk will depend on how much tissue we received.
Chunk for RNA. Place in 1.5 ml cryovial containing 1 mL RNALater and store overnight at room temp. The next day, drain out RNALater and snap freeze in liquid nitrogen. After sample is frozen, store at -80º.C Size of chunk will depend on how much tissue we received.
Chunk for viable freezing for possible future PDX generation. Cut viable tissue into small squares ~1.5 mm x 1.5 mm. Place a minimum of 12 chunks into 1 mL of freezing media (10% DMSO, 50% FBS, 40% RPMI-1640 or DMEM) in a cryovial and place in styrofoam or Mr. Freezy container and place in -80ºC freezer for controlled freezing.
The following procedure is based on the Miltenyi MACs dissociation protocol 2.2.1 "Dissociation of soft tumors".
Prepare enzyme solution in MACs C tube:
4.7 ml RPMI
200 µl H enzyme
100 µl R enzyme
25 µl A enzyme
Dice the sample using scissors and/or scalpel in petri dish on ice and transfer pieces to the MACs C tube containing the enzyme solution. Shake to make sure pieces are not stuck in the teeth of the tube.
Place MACs C tube in tissue dissociator and run the h_tumor_01 program, which takes ~ 30 seconds.
Transfer MACs C tube to a rotator in a37ºC incubator and incubate for 30 minutes.
Place MACs C tube in tissue dissociator and run the h_tumor_02 program, which takes ~30 seconds.
Transfer MACs C tube to a rotator in a37ºC incubator and incubate for 30 minutes.
Place MACs C tube in tissue dissociator and run the h_tumor_03 program, which takes ~30 seconds.
Optional: Centrifuge the MACs C tube for 30 seconds at 300g to collect sample material at bottom
Filter the contents of the MACs C tube using a 70 µm strainer into a 50 ml conical. It is possible to do this by pipetting through the septum-sealed opening in the center of the C tube cap. Apply an additional 20 mL of RPMI-1640 to rinse the strainer.
Centrifuge cell suspension for 7 minutes at 300g at 25ºC to pellet the cells. Remove and discard supernatant.
Resuspend in 500 µl of RPMI-1640 or hypothermosol and transfer to a 1.5 mL microcentrifuge tube.
Optional: If cell pellet is red, that indicates a large number of red blood cells. To lyse the red blood cells add 1 mL of red blood cell lysis buffer at 25ºC. Mix by inverting (do not vortex) and rock the tube on a gyrator for 10 minutes at 25ºC. Centrifuge for 5 minutes at 500g at 25ºC. Remove and discard supernatant. Resuspend cell pellet in 500 µl of hypothermosol.
Remove a 10 µl aliquot of cells and count using trypan blue or an automated counter like Cell Countess
Count 1: Live cells/µl: ________________________, Dead cells/µl: ________________________
Count 2: Live cells/µl: ________________________, Dead cells/µl: __________________________
Total volume of cells (µl): ______________________
Prepare an aliquot to deliver to BMGC sequencing facility. Provide 500 µ l at a concentration of 1,000 live cells/µl in hypothermosol (500,000 cells total).
Live cells/µl _____________ / 500,000 = ___________________ µl. Add ____________µl to bring to 500 µl
Transport microcentrifuge tube with cells to sequencing facility.
Remaining cells can be plated in media to attempt to grow out cell lines. Alternatively, use the remaining cells for flow cytometry or cytof.