Genome-wide CRISPR-Cas9 screen

Genome-wide CRISPR-Cas9 screen：

1, Seed Cas9 stably expressed iBMDMs at 2×10⁶ cells/dish in DMEM medium without antibiotics in 10 cm dish (30-35 dishes);

2, Infect the cells with lentivirus-containing sgRNA library at a multiplicity of infection (MOI) of 0.3 in DMEM medium containing 1 ng/µl polybrene (without antibiotics);

3, Change the medium at 24 h after infection;

4, Sixty hours later, cells were treated with 5 ng/µl puromycin to remove uninfected cells;

5, Change the medium every 24 h, and keep puromycin treatment for three days;

6, Six days after puromycin treatment, the transduced cells were seeded in 40 × 10 cm dishes (8 × 10⁶ cells/dish) and treated with 10 ng/ml LPS plus 125 nM 5z7 for 6 hours (3 × 10⁸ transduced cells were collected as the control sample);

7, Three days later, the surviving cells were reseeded and treated with 10 ng/ml LPS plus 250 nM 5z7 overnight;

8, Two days later, surviving cells were again treated with 20 ng/ml LPS plus 250 nM 5z7 overnight before surviving cells were harvested.

DNA extraction：

1, Surviving cells and control sample were lysed in the SNET buffer (20 mM Tris-HCl pH 8.0, 5 mM EDTA, 400 mM NaCl, 1% SDS and 400 mg/ml Proteinase K), 3×10⁸ cell/5 ml buffer;

2, Incubate at 65℃, 2-3 h, until the sample is clear;

3, Add an equal volume of PCI (phenol/chloroform/isoamyl alcohol, 25:24:1, pH>7.8), mix and microcentrifuge 10 min at 12,000 rpm at room temperature;

4, Carefully transfer the top (aqueous) phase to a new tube;

5, repeat steps 3-4;

6, Add an equal volume of CI (chloroform/isoamyl alcohol 24:1), mix and centrifuge for 10 min at 12,000 rpm;

7, Carefully transfer the top (aqueous) phase to a new tube;

8, Add 2.5 vol of ice-cold 100% ethanol. Mix and place in -70℃ overnight;

9, Spin 20 min at 14,000 rpm at 4℃ and remove the supernatant;

10, Carefully add 1 ml cold 70% ethanol to wash the DNA pellets;

11, Spin 10 min at 12,000 rpm at 4℃ and remove the supernatant;

12, Air dry the pellet at room temperature;

13, Dissolve the DNA in H₂O (0.5 µg/µl). Larger quantities of DNA may require vortexing and brief heating (5 min at 65 ℃) to resuspend.

Next generation sequencing：

Genomic DNAs were amplified by two step PCR using the 2× Hieff Canace Gold PCR Master Mix:

1, Set up PCR reactions with 10 μg of genomic DNA in 100 μL final volume using 50 μL of 2× Hieff Canace Gold PCR Master Mix, and 2 μL each of 10 μM primers (Forward primer: 5’-AAT GGA CTA TCA TAT GCT TAC CGT AAC TTG AAA GTA TTT CG-3’, Reverse primer: 5’-TCT ACT ATT CTT TCC CCT GCA CTG T-3’);

2, Perform as many PCR replicates per sample as needed to maintain desired library coverage. For control sample, 30 PCR replicates were necessary, while 3 PCR replicates were sufficient for screening samples;

3, The following thermocycler conditions should be used: 98 ℃ for 30 S, 18×(98 ℃ for 10 s, 58 ℃ for 20 s, 72 ℃ for 25 s), 72 ℃ for 5 min;

4, Pool all primary PCR products for each sample.

5, Set up secondary PCR reactions using 100 μL final volume, 50 μL of 2× Hieff Canace Gold PCR Master Mix, 10 μL of the pooled primary PCR product and 2 μL each of 10 μM Illumina sequencing primers(1), 7 PCR replicates for each sample;

6, The following thermocycler conditions should be used: 98 ℃ for 30 S, 20×(98 ℃ for 10 s, 63 ℃ for 20 s, 72 ℃ for 10 s), 72 ℃ for 5 min;

7, Load samples on a 2% agarose Gel, and isolate the secondary PCR product at 260-270 bp;

8, Proceed with Next generation sequencing on an Illumina HiSeq 2500 sequencer.

1.         Joung, J., Konermann, S., Gootenberg, J. S., Abudayyeh, O. O., Platt, R. J., Brigham, M. D., Sanjana, N. E., and Zhang, F. (2017) Genome-scale CRISPR-Cas9 knockout and transcriptional activation screening. Nat Protoc 12, 828-863