GSM6304116: microglia CRISPRi-hypr-seq screen rep2; Homo sapiens; OTHER
GSM6304116
The HyPRseq experiment was performed according to the published protocol (33376219). Briefly, cells were fixed in 4% formaldehyde solution and permeabilized in 70% ice cold ethanol. 4 pairs of ssDNA probes for each target gene were designed using HyPR-seq pipeline (https://github.com/EngreitzLab/hypr-seq). All probes were pooled at 20nM and incubated overnight at 37. After hybridization cells were washed and sequentially incubated with B1H1 hairpin (HCR tech), readout oligo, T4 ligase at 37°C for one hour individually. Following ligation, cells were resuspended in PBST and filtered through a 20 μm pluriStrainer. Single cell suspension was checked under microscope and counted for droplet generation. To generate emulsions we first combined 1× EvaGreen Supermix (Bio-Rad Cat #186- 4033), 500 nM indexing primer, 2,000 cells and 20,000 barcoded beads (Chemgenes, as described above) in a 20 μLreaction. Once the PCR mix is made, emulsions were generated using QX200 Digital Droplet Generator (Bio-Rad #1864002) as per manufacturer's instructions. After the droplets were generated, emulsions were gently transferred into PCR tubes and placed on ice and under the UV lamp (6.5 J/cm2 at 365 nm) about 3-8 cm away from the bulb for 5 mins. After UV exposure, the following cycling conditions were used for droplet PCR amplification: denaturation: 94°C for 30 sec; cycling: 25 cycles of 94°C for 5 sec, 64°C for 30 sec, and 72°C for 30 sec; final extension: 72°C for 5 mins. All PCR cycling steps were performed with 50% ramp rate (2 °C/min).To break down emulsions, PCR product were combined and half volume of 97% 1H,1H,2H,2H-Perfluoro-1-octanol (Sigma-Aldrich #370533) was added. Then tubes were vortexed for 5 sec and spun at 1000 g for 1 min. The top aqueous layer was carefully removed and cleaned with 1.8X SPRI beads according to manufacturer's instructions. The amplicon libraries were sequenced with customer primers in deep sequencing. For each replicate we loaded ~50k cells in the emulsion generation step and ~15k cells could be packaged into droplets according to the Poisson distribution. We sequenced ~5000 reads/cell covering 18 target genes and 28 gRNA barcode sequences.