protocol_173.txt

Re-amplification of CRISPRa and CRISPRi libraries
Dilute each sub-library to 50 ng/ul in water or EB Electroporate the library.
Pre-chill 0.1 cm cuvettes, megaX cells, 10% glycerol on ice.
Follow the table for the amounts of sub-library plasmid DNA and MegaX competent cells, mix gently and incubate on ice for 30 min.  
Add pre-chilled 10% glycerol to the MageX-library mix for a final 75 ul, transfer the mix to a prechilled 0.1 cm cuvette.
Electroporate at 2.0 kV, 200 ohms, 25 uF (Gene Pulser Xcell, Bio-rad).  
Transfer cells to a culture tube.  
Use 1 ml pipettes and gel loading tips.
Wash cells out gently with 300 ul S.O.C. twice (total 600 ul).
Incubate at 37oC, 250 rpm, 1.5 hour.
Plate the transformations.
Plate all in one large square plate per sub-library, use autoclaved beads.
Incubate at 37oC for 18 hours.
Collect all colonies with LB and do one maxiprep per plate, elute in 500 ul EB; an ideal concentration is about 2~3 ug/ul.
To sequence the library, you can PCR the sgRNA region with the following primers (a common 3’ primer, with 2 different 5’ primers for CRISPRa or CRISPRi).
CRISPRi TSS common 3' caagcagaagacggcatacgaCGACTCGGTGCCACTTTTTC.
oCRISPRi TSS_1 (TruSeq Index 12 CTTGTA), aatgatacggcgaccaccgaGATCGGAAGAGCACACGTCTGAACTCCAGTCACCTTGTAgcacaaaaggaaactcaccct.
oCRISPRi TSS_2 (TruSeq Index 6 GCCAAT) aatgatacggcgaccaccgaGATCGGAAGAGCACACGTCTGAACTCCAGTCACGCCAATgcacaaaaggaaactcaccct
Pool sub-libraries proportionally (based on the number of sgRNAs) to have the CRISPRa or CRISPRi library, measure the pooled concentration and dilute it to 400 ng/ul for PCR
Run 3 tubes of 100 ul PCR reactions