P27-87
Mus musculus
Protocols: B16-F10 melanoma model: The C57BL/6 derived B16-F10 melanoma cell line was purchased from American Type Culture Collection (ATCC) and cultured in Dulbecco's Modified Eagle medium (DMEM, Life Technologies), supplemented with Penstrep and 10% FBS. 2.5 x105 B16-F10 cells were injected subcutaneously into the shoulders of Cyp11a1-mCherry mice. After 12 days animals were sacrificed and tumour tissues were collected for analysis. All tumours were below 500mm3 volume.Tumor Tissue Processing Tumors were mechanically dissociated and digested in 1mg/ml collagenase D (Roche), 1mg/ml collagenase A (Roche) and 0.4mg/ml DNase I (Sigma) in IMDM media containing 10% FBS, at 37°C for 40 mins. EDTA was added to all samples to neutralize collagenase activity (final concentration 5mM) and digested tissues were passed through 70μm filters (Falcon). Cell sorting Once processed, single cell suspension tumour samples were incubated with a fixable fluorescent viability stain (Life Technologies) for 20mins (diluted 1:1000 in PBS) prior to incubation with conjugated primary antibodies for 30 mins at 40C. Antibodies were diluted in PBS 0.5% BSA. Stained samples were sorted using the BD Influx cytometer system. cDNA preparation: Plate based scRNA-seq was performed with the NEBNext Single Cell/Low Input RNA Library Prep Kit for Illumina (New England Biolabs Inc, E6420L). Briefly, single cells were sorted into a pre-prepared 96-well plate (Eppendorf, Cat. No. 0030128648) containing 2 µl of 1X NEBNext Cell Lysis Buffer. Sorted single cells were sealed and spun at 100 x g for 1 minute then immediately frozen on dry ice and stored at -80 °C. cDNA generation was then performed in an automated manner on the Agilent Bravo NGS workstation (Agilent Technologies). Briefly, 1.6 µl of Single Cell RT Primer Mix was added to each well and annealed on a PCR machine (MJ Research Peltier Thermal Cycler) at 70°C for 5 minutes. 4.4 µl of Reverse Transcription (RT) mix was added to the mixture and further incubated at 42°C for 90 minutes followed by 70°C for 10 minutes to generate cDNA. 32 µl of cDNA amplification mix containing NEBNext Single Cell cDNA PCR MasterMix and PCR primer was mixed with the cDNA, sealed and spun at 100 x g for 1 minute. cDNA amplification was then performed on a PCR machine (MJ Research Peltier Thermal Cycler) with 98°C 45 s, 20 cycles of [98°C 10 s, 62°C 15 s, 72°C 3 mins], 72°C 5 mins. The 96-well plate containing the amplified cDNA was purified with an AMPure XP workflow (Beckman Coulter, Cat No. A63880) and quantified with the Accuclear Ultra High Sensitivity dsDNA kit (Biotium, Cat. No. 31028). ~10 ng of cDNA was stamped into a fresh 96-well plate (Eppendorf, Cat. No. 0030128648) for sequencing library preparation. Sequencing libraries were then generated on the Agilent Bravo NGS workstation (Agilent Technologies). Purified cDNA was fragmented by the addition of 0.8 µl of NEBNext Ultra II FS Enzyme Mix and 2.8 µl of NEBNext Ultra II FS Reaction buffer to each well and incubated on a PCR machine (MJ Research Peltier Thermal Cycler) 72°C 15 mins, 65°C 30 mins. A ligation mixture was then prepared containing NEBNext Ultra II Ligation Master Mix, NEBNext Ligation Enhancer and 100 µM Illumina compatible adapters (Integrated DNA Technologies) and 13.4 µl added to each well of the 96-well plate. The ligation reaction was incubated on the Agilent workstation at 20°C for 15 minutes and then purified and size selected with an AMPure XP workflow (Beckman Coulter, Cat No. A63880). 25 µl of KAPA HiFi HS Ready Mix (Kapa Biosystems, Cat. No. 07958927001) was then added to a pre-prepared 96-well plate (Eppendorf, Cat. No. 0030128648) containing 100 µM i5 and i7 indexing primer mix (50 µM each) (Integrated DNA Technologies). The indexing primers pairs were unique to allow multiplexing of up to 384 (4x 96-well plates) cells into one sequencing pool. The plate containing the PCR Master Mix and indexing primers was stamped onto the adapter ligated purified cDNA, sealed and spun at 100 x g for 1 minute. Amplification was performed on a PCR machine (MJ Research Peltier Thermal Cycler) with 95°C 5 min, 8 cycles of [98°C 30 s, 65°C 30 s, 72°C 1 min], 72°C 5 mins. The PCR products were pooled in equal volume on the Biomek NXP automated liquid handler (Beckman Coulter) and the pool purified and size selected with an AMPure XP workflow (Beckman Coulter, Cat No. A63880). The purified pool was quantified on an Agilent Bioanalyser and combined with 3 additional 96 plex pools to produce a sequencing library containing 384 cells, and sequenced on one lane of an Illumina HiSeq 4000 instrument.