CRISPR 128 genomic zebrafish loci – sgRNAs – Pull-down expt – Canonical – Mutant       pool1
Total RNA was isolated from embryos injected with sgRNAs using TRIzol reagent (Life Technologies). Purified total RNA was then subjected to reverse transcription using the SuperScript III First Strand Kit (Invitrogen), following manufacturer’s protocol, and a primer containing a 3’ sequence complementary to the constant part of the sgRNAs and a 5’ sequence corresponding to part of the Illumina TruSeq Adapter (5’- AGACGTGTGCTCTTCCGATCTCGGACTAGCCTTATTTTAACTTGC-3’). The resulting first-strand cDNAs were purified using Agencourt AMPure XP system (Beckman Coulter) following manufacturer’s protocol. cDNAs were dissolved in 10 µL of water and a ssDNA linker was added at their 3’ ends (5'-/5Phos/NNNNGATCGTCGGACTGTAGAACTCTGAAC/3InvdT/-3’, where /5Phos/ is a 5’ phosphate and /3InvdT/ is an inverted deoxythymidine) using CircLigase ssDNA Ligase (Epicentre), with slight modifications to the manufacturer’s protocol. In brief, reagents were added to 3 µL of dissolved cDNA samples to reach the following final concentrations in a total volume of 10 µL: 1x CircLigase buffer, 0.05 mM ATP, 2.5 mM MnCl2, 10% PEG 6000, 1M betaine, 5 µM ssDNA linker and 50 U CircLigase enzyme. Ligation mixture was then incubated at 60˚C for 2 h, 68˚C for 1 h and 80˚C for 10 min to deactivate the CircLigase. Volume was increased by the addition of 10 µL of water and ligated products were purified using Agencourt AMPure XP system (Beckman Coulter) following manufacturer’s protocol. PCR amplification was performed on the ligated product using Illumina primers (Illumina Small RNA forward primer, 5’- AATGATACGGCGACCACCGACAGGTTCAGAGTTCTACAGTCCGA-3’; Illumina TruSeq reverse index, 5’- CAAGCAGAAGACGGCATACGAGATxxxxxxGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3’; indexes 6, 7, 12-16, 19, 23, 25, 27 were used in this study). PCR products were purified on an 8% native polyacrylamide gel and bands corresponding to sgRNA final library size (179 nt) were extracted. DNA was eluted, ethanol precipitated, dissolved in water and sent for sequencing.  For input samples, in vitro transcribed sgRNA cocktails used for injection were pooled together, diluted 1/100 and 1 µL of this dilution was combined with total RNA isolated from uninjected embryos prior to reverse transcription. Alternative sgRNA samples and RNA samples isolated from the Cas9 pull-down experiment were treated as mentioned above with few modifications. Briefly, 50 U of RNase I was added during the RNase H treatment prior to AMPure XP purification. Finally, the AMPure XP purification previous to the PCR amplification was substituted by a 10% urea-denaturing PAGE purification step. Bands corresponding to the ligated product size (114 nt) were cut. DNA was then eluted, ethanol precipitated, dissolved in water and PCR amplified as mentioned above.
Yale University
crispr_rules_sgrna_can_pd_mut3_AG00934