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
