GSM4783951: K562_timecourse_2hr_3; Homo sapiens; OTHER
For HyPR-Seq, cells are harvested in 1X phosphate buffered saline (PBS) at 350g for 5 mins at 4°C in a swinging bucket rotor. Cells were fixed in 4% Formaldehyde solution (4% formaldehyde in 1XPBS and 0.1% Tween 20) for one hour at room temperature at a concentration of 1 million cells per ml. The fixed cells are then washed twice with of 1X PBS containing 0.2% Tween 20 (1X PBST) at 850g for 5 mins at 4°C. The washed cells were then permeabilized in 70% ice cold ethanol at a concentration of 1 million cells per ml and stored at 4°C for 10 mins. The permeabilized cells are then harvested and washed twice with 1XPBST (850g for 5 mins). The cells were then transferred to 2ml V-bottom tubes for subsequent probe hybridization with up to 5 million (cells per hybridization reaction. The cells were resuspended in 0.5mls of probe hybridization buffer (5X SSC, 30% formamide, 0.1% Tween 20) and was incubated at 37°C for 5 min. Probes mix was prepared at a concentration of 20nM per probe in 2mls of probe hybridization buffer. The prehybridized cells are harvested at 850g for 5 mins at room temperature, resuspended in the probes mix and incubated overnight at 37°C in a hybridization oven (VWR Cat# 10055-006). The probe hybridized cells were harvested (850g for 5 mins) and washed with probe hybridization buffer with a 10min incubation of cells in buffer at 37oC before the spin. These wash and incubation steps were done a total of four times. After the last spin, the cells were incubated in a 75nM H1 hairpin mix. To prepare the H1 hairpin mix for one hybridization reaction, 5ul of 3uM H1 hairpin was snap cooled (heat at 95°C for 90 seconds and cooled to room temperature for 30 min) and resuspended in 200ul of 5XSSCT buffer (5XSSC and 0.1% Tween 20). The cells were resuspended in 200ul of H1 hairpin mix was incubated at 37°C for one hour in the hybridization oven. After the H1 hairpin hybridization, the cells are washed twice with 5XSSCT at room temperature (850g for 5 mins). The cells are then resuspended in a 75nM “readout” oligo mix. To prepare the “readout” mix for one hybridization reaction, 5ul of 3uM of “readout” oligo was snap cooled as detailed above and resuspended in 200ul of 5XSSCT buffer. The cells are resuspended in the oligo mix and incubated 37°C for one hour. After oligo incubation, the cells were harvested and washed in 5X SSCT three times (850g for 5 mins). After the last spin the cells were suspended in and washed 1X T4 Ligase reaction buffer (NEB #B0202S). Finally, the cells were resuspended in Ligase buffer (1X T4 ligase buffer, 1:50 dilution of T4 DNA ligase NEB #M0202S) and incubated at room temperature for one hour. Following this final incubation, cells were washed three times in 1XPBST and filtered through a 20micron filter (20um pluriStrainer cat. 43-0020-01). The cells were then counted using a hemocytometer (Fisher Scientific SKU#DHCF015) and checked for single cell suspension before proceeding to droplet generation. For long term storage of the cells, add RNase inhibitor to the cell suspension (NEB M0314S). To generate emulsions, we first combined 2X Evagreen supermix (Biorad cat 186-4033), 500nM indexing primer (Table S3), 2,000 cells and 20,000 barcoded beads (Chemgenes) in a total of 20ul per reaction. Once the PCR mix is made, emulsions were generated using QX200 Digital Droplet Generator (BioRad #1864002) as per manufacturer's instructions. Briefly, the droplet generation cartridge (Biorad #186-4007) was inserted into the holder (Biorad 186-3051) and 20ul of the prepared PCR mix was added to the middle sample wells as per instructions. 70ul of the droplet generating oil (Biorad, 186-4005) was added into the bottom row and the gasket (Biorad 186-4007) was placed over the filled cartridge and placed in the droplet generator. Once the droplets were formed, the cartridge containing the emulsions was placed under the UV lamp (6.5 J/cm2 at 365 nm) about 3-8cms away from the bulb for 5min. After UV exposure, ~50ul of droplets per well were transferred to 96 well plates (Eppendorf 951020362) and sealed with foil (Biorad #181-4040) using a plate sealer (PX1 Plate Sealer 181-4000) for droplet PCR amplification (Eppendorf Mastercycler Pro E90030010). The following cycling conditions were used: Denaturation: 94°C for 30 sec; Cycling: 30 cycles of 94°C for 5 sec, 64°C for 30 sec, and 72°C for 30 sec; Final extension: 72°C for 5 min. All PCR cycling steps were performed with 50% ramp rate (2°C/min). To break the emulsions, one to four PCR wells were combined in a tube and equal volumes of 100% PFO were added (~150l). The tubes were vortexed for 5 sec to ensure complete emulsion breakage and spun at 1000g for 1min. The top aqueous layer was carefully removed and cleaned with 1.8X SPRI beads according to manufacturer's instructions. The amplicon libraries were loaded on a gel to determine size (206bp) and to ensure no primer dimers remained. The libraries were quantified by Qubit before proceeding to sequencing.