Alice:Single cell tracing and RNA-seq Lab Notes/Belmonte lab collaboration projects

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In vivo genome editing via CRISPR-Cas9 mediated homology-independent targeted integration[edit]

project overview[edit]

  • CRISPR mediated tissue specific knock-in of GFP into the Rosa-26 locus transgenic mice, the locus is driven by CAG promoter
  • Kei from Belmonte lab has already established the system and injected into transgenic mice muscle cells, also heart and liver
  • we will perform analysis on DNA-seq validation/quantification of knock-in efficiency and off-target rate/sites

data for revision of the manuscript[edit]

  • The sequencing are done in SE 150+8+8 bp setting
  • The sample were pooled together as following, and sequenced on 5/2/2016 Miseq:
2nd round of primer 1 (i7) 2nd round of primer 2 (i5) conc. (ng/ul) vol amount total vol total amount final concentration
1 On target/Liver Cas- N701 S501 11.2 4.5 50 67.5 800 11.85
2 On target/Liver Cas+ N701 S502 13.9 3.6 50
3 On target/Heart Cas- N701 S503 11.2 4.5 50
4 On target/Heart Cas+ N701 S504 8.2 6.1 50
5 LAMPCR-GFP3'/Liver Cas- N701 S505 11.5 4.3 50
6 LAMPCR-GFP3'/Liver Cas+ N701 S506 12.7 3.9 50
7 LAMPCR-GFP3'/Heart Cas- N701 S507 9.5 5.3 50
8 LAMPCR-GFP3'/Heart Cas+ N701 S508 7.4 6.8 50
9 LAMPCR-AAVITR5'/Liver Cas- N702 S501 12 4.2 50
10 LAMPCR-AAVITR5'/Liver Cas+ N702 S502 16 3.1 50
11 LAMPCR-AAVITR5'/Heart Cas- N702 S503 12.8 3.9 50
12 LAMPCR-AAVITR5'/Heart Cas+ N702 S504 13.7 3.6 50
13 LAMPCR-AAVITR3'/Liver Cas- N702 S505 13.1 3.8 50
14 LAMPCR-AAVITR3'/Liver Cas+ N702 S506 15 3.3 50
15 LAMPCR-AAVITR3'/Heart Cas- N702 S507 16.3 3.1 50
16 LAMPCR-AAVITR3'/Heart Cas+ N702 S508 14.2 3.5 50

Analysis[edit]

  • adopt an different published approach to analyze the OTS and on-target data from first batch of cells, so these are the papers that I looked at:
    • Genome editing assessment using CRISPR Genome Analyzer (CRISPR-GA). Bioinformatics. 2014 Oct 15;30(20):2968-70. doi: 10.1093/bioinformatics/btu427. Epub 2014 Jul 1. (http://54.80.152.219/index.php)
    • DNA targeting specificity of RNA-guided Cas9 nucleases. Nat Biotechnol. 2013 Sep;31(9):827-32. doi: 10.1038/nbt.2647. Epub 2013 Jul 21.
Workflow of the analysis (both from above paper and ourselves')
1. only uses aligners of choice (e.g. BLAT or Smith-Waterman alignments) to perform sequence alignment against custom Ai14 transgenic mice genome
chr6 tdTomato knock-in: 113076031-113077114 (left arm); 113077114- 113076032(right arm)
2. then count the # of reads that fulfill certain criteria (e.g. matched base­ pairs comprised more than 85% of their total length)
3. calculate the indel rate by having # of reads with indel/total reads(# of reads with indel+# of reads with ~95% of match)
4. perform MLE analysis between negative control and positive sample to calculate the estimated indel rate

Alignment (perl script):
my @file=glob("*CAGOn*.fastq")
/home/k4zhang/softwares/bwa-0.7.5a/bwa mem -O 4 -R \'\@RG\tID:$file\tSM:OTS\tPL:illumina\tLB:$file\' /home/zhl002/genomeDB/Ai14/mm9_Ai14.fa $file > $file.sam
samtools view -Sb -o $file.bam $file.sam
samtools sort -o $file.sorted.bam $file.bam
samtools index $file.sorted.bam
Read filtering (perl script):
use strict;
my $config=shift @ARGV;
my $minM=127;
open F,"$config";
while(<F>){
chomp;
my($cor,$file)=split /\s+/;
next if /^\s+$/;
chdir "/oasis/tscc/scratch/zhl002/kei_012616/data";
my @bam=glob("*$file*Ai14_sorted*bam");
foreach my $bam(@bam){
system("samtools view $bam $cor> $bam.pad");
open F2,"$bam.pad" || die "cannot open $bam.pad\n";
my $indelReads;
my $totalReads;
my $indelNum;
my $SHclipReads;
my $qname;
my $MreadsNum;
my $NskipReads;
my $quitematch;
while(<F2>){
$totalReads++;
my @tmp=split /\s+/;
my @M=($tmp[5]=~/(\d+)M/g);
my $Mnum=&msum($tmp[5]);
my $maxMnum=&max(@M);
if($maxMnum>=$minM){
$MreadsNum++;
$indelReads++ if $tmp[5]=~/(\d+I)|(\d+D)/;
$indelNum +=(&INSsum($tmp[5])+&DELsum($tmp[5]));
$NskipReads++ if $tmp[5]=~/(\d+N)/;
$quitematch ++ if $Mnum>=140;
$SHclipReads++ if $tmp[5]=~/(\d+S)|(\d+H)/;
}
}
my $ratio1=sprintf("%.4f",$indelReads/($quitematch+$indelReads+1));
my $ratio2=sprintf("%.4f",$indelNum/($quitematch+$indelReads+1));
my ($id,undef)=split /-/,$bam;
print "$bam\t$file\t$id\t$cor\t$indelReads\t$indelNum\t$quitematch\t$ratio1\t$ratio2\t$SHclipReads\t$totalReads\n";
close OUT;
close OUT2;
system("rm $bam.pad");
}
}

sub msum($){
my $string=shift @_;
my @num=($string=~/(\d+)M/g);
my $sum;
foreach my $num(@num){
$sum +=$num;
}
return($sum);
}
sub INSsum($){
my $string=shift @_;
my @num=($string=~/(\d+)I/g);
my $sum;
foreach my $num(@num){
$sum +=$num;
}
return($sum);
}

sub DELsum($){
my $string=shift @_;
my @num=($string=~/(\d+)D/g);
my $sum;
foreach my $num(@num){
$sum +=$num;
}
return($sum);
}

sub max(@){
my @xx=@_;
my $max=0;
foreach my $x(@xx){
$max=$x if $x>$max;
}
return($max);
}
MLE algorithm (R script): 
#n is the number of reads with in/del
n=36884 
#R is the total number of reads (reads with indel + reads with perfect match of full sequence length, here is 150bp)
R=564402
#p is the indel ratio (reads with indel/total reads) in positive sample (cas+)
p=0.0755	
#q is the indel ratio (reads with indel/total reads) in negative/control sample (cas-, but donor plasmid is present)
q=0.0596
qqq<-c()
#OPTIONAL: use input file that has list of samples with the above values printed in each column, then we can perform MLE on multiple samples at once
proxy<-read.table("proxy.txt",head=F,sep="\t",row.names=1,as.is=T)
qqq<-c()
for (i in 1:nrow(proxy)){
 n=proxy[i,1]
 R=proxy[i,2]+n
 p0=proxy[i,3]
 q0=proxy[i,4]
 pp<-c()
 qq<-c()
 for(p0 in seq(p0,0,-0.00001)){
   q<-dbinom(round(n-R*p0),round(R*(1-p0)), q0)
   q1=c(q,p0)
   qq<-rbind(qq,q1)
 }
 qx<-qq[which.max(qq[,1]),]
 qqq<-rbind(qqq,qx)
}
rownames(qqq)=rownames(proxy)
qqq
write.table(qqq[,2]*100,file="name.txt",row.names=T,col.names=NA,sep="\t",quote=F)
LAMPCR on-target ratio analysis:
perl script for read filtering:
#!/usr/bin/perl
use Cwd;
chdir getcwd;
my $bam=shift @ARGV;
system("samtools view $bam $cor> $bam.pad");
open F2,"$bam.pad";
open OUT, ">$bam.stat";
my %qreads;
while(<F2>){
my @tmp=split /\s+/;
next if $tmp[5]=~/\*|uncut/i;
if($tmp[5]=~/(\d+)S\w+(\d+)S|(\d+)S\w+(\d+)H|(\d+)H\w+(\d+)S|(\d+)H\w+(\d+)H/){
my $num=$1+$2+$3+$4+$5+$6+$7+$8;
}
my $solid=151-$num;
$qreads{$solid}++;
}

foreach my $num(sort keys %qreads){
       print "$num\t$qreads{$num}\n";
} 
knock-in ratio calculation R script:
j=0.15
k=0.5
rlt<-c()
file=list.files(pattern="*sam.tam.stat.output")
for (i in 1:length(file)){
data=read.table(file[i],sep="\t")
sub1<-subset(data,data[,1]<151*j)
sub2<-subset(data,data[,1]<151*k)
n=sum(sub1[,2])
R=(sum(sub2[,2]))
ratio<-sum(sub1[,2])/(sum(sub2[,2]))
tmp<-c(n,R,ratio)
rlt<-rbind(rlt,tmp)
}
rownames(rlt)=file
write.table(rlt,file="ratio.rlt.txt",row.names=T,col.names=NA,sep="\t",quote=F)

Results[edit]

# chromosome position sequence PAM % indel (MLE)
On_Target chr6 GAGGAACTTCTTAGGGCCCGCGG CGG 18.917
On_Target_Liver Chr6 GAGGAACTTCTTAGGGCCCGCGG CGG 11.477
On_Target_Heart Chr6 GAGGAACTTCTTAGGGCCCGCGG CGG 3.301
OTS1 chr13 9534141 CAGGAACTTCTTAGGTCCCGTGG TGG 0.0001
OTS2 chr14 85157555 CGGAAACATCTTAGGGCCCGGGG GGG 0.0001
OTS3 chr2 74925639 GGAGTACTTCTTAGGGCCCACAG CAG 0.14
OTS4 chr15 69159526 CAGGAACATATTAGGGCCCAGGG GGG 0.0001
OTS5 chr12 100704049 CAGGAACATGTTAGGGTCCGAGG AGG 0.215
OTS6 chrX 91969928 CAGGAACGTGTTCGGGCCCGCGG CGG 0.0001
OTS7 chrX 92043584 CAGGAACGTGTTCGGGCCCGCGG CGG 0.0001
OTS8 chr10 17440682 CAGGAACTTCTTAGTGCCCTAAG AAG 0.004
OTS9 chr18 25702486 GTGGACCTTTTCAGGGCCCGTGG TGG 0.784
OTS10 chr18 9212447 GAGGCCCCTCTTCGGGCCCGGAG GAG 0.388
OTS11 chr14 46862821 GTGGAGCATCTTAGGGCCAGTGG TGG 0.0001
OTS12 chr12 31652981 GAGCAACAACTTAGGGCCTGCAG CAG 0.197
Sample ID % On-target (MLE)
LAMPCR_AAVITR3_Liver 7.6411176
LAMPCR_AAVITR3_Heart 3.072334
LAMPCR_GFP3_Liver 6.0686491
LAMPCR_GFP3_Heart 1.1098576

data for initial submission of the manuscript[edit]

  • Kei finished locus specific PCR using the following 12 pairs of primers listed below:
CAGon-F
TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGTCTAGAGCCTCTGCTAACCATGTTCA
CAGon-R
GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGAGATCTGCAAGCTTTCATTTATTCATCGC
61℃->68℃  245bp (+KI 1720bp)
CAG-OTS1-F
TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGAGGGTAGATAACATCTTATTACAATGAGT
CAG-OTS1-R
GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGTACAATAATTTAGTGCCACCAGATGGAA
60℃->68℃  171bp  F
CAG-OTS2-F 
TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCAGGAGATTGAGGAACCCCAT
CAG-OTS2-R
GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGCCACCATCCTCCCCAACCAG
60℃->68℃  265bp  F
CAG-OTS3-F
TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGATTTCACTGCCCATAGTTTTTATTAAACG
CAG-OTS3-R
GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGTTTAACAGGAGGAAATGTTGCCCTT
60℃->68℃  197bp  F
CAG-OTS4-F
TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGAACCAGACAAATGGGCATATAGGAA
CAG-OTS4-R
GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGTGGCCTGCTCAGTATTTTATATCCAGA
60℃->68℃  207bp  F
CAG-OTS5-F
TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGTAGGGCAGCTGAGAATAATGTAAAAGTTCG
CAG-OTS5-R
GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGGCAAACCTTAAAAAGCATCAGA
60 (56)℃->68℃  142bp  F (reverse design)
OTS6 and OTS7 have the same sequence region:
CAG-OTS6/7-F2
TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCGCGGTCCCAGCATATTTAAATCACGG
CAG-OTS6/7-R2
GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGTGGGGGCCTGACAAGCCAGA
63℃->68℃  297bp  F (reverse design)
CAG-OTS8-F
TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGACTAGTTTACTATGCCCGCTGA
CAG-OTS8-R
GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGCCACTTGCCCATCATCCAC
60℃->68℃  279bp  F
CAG-OTS9-F
TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGTTCTTCTCAACAAGCTCGCCCCTTT
CAG-OTS9-R
GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGTTTCGCTTCAGCTTCGCATACCCT
60℃->68℃  141bp  F (reverse design)
CAG-OTS10-F
TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGCGGCAGATACAAGAGGACCGAC
CAG-OTS10-R
GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGCTCATCCTCCCGAGTTCGTGTAGGAC
60 (63)℃->68℃  285bp  F
CAG-OTS11-F
TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGTGCAGGCTATAAATCCAAATTCCC
CAG-OTS11-R
GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGCCTTTGGAATTATACATTTCCCGTCT
60℃->68℃  202bp  F
CAG-OTS12-F
TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGAAAGGCCAGATCACCAGAGAATACCAG
CAG-OTS12-R
GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGACCAAGGGGAATGAATGTCT
60℃->68℃  284bp  F
  • 1st round of PCR (amplification of the target genome locus with overhang tag
Total PCR reaction: 50 ul
Template genomic DNA in each reaction: 55.2 ng
   PCR cycle:  35 cycle
      ↓
   Purification by AMpure XP and resuspend in 40ul water
↓
2nd round of PCR (Amplification of the 1st round PCR product with Nextera Index primer 1 (N7xx) and primer 2 (S5xx))
  Total PCR reaction: 50 ul
  Template DNA in each reaction: 2ul of the purified PCR product from 1st round PCR
   PCR cycle:  10 cycle
     ↓
   Purification by AMpure XP and resuspend in 40ul water
  • The samples submitted for Miseq on 1/26/2016

ZL-kei-OTSPCR-012416 (pooled all 24 libraries from below); SE 150+8+8; ~300 size range; Read 1 primer: HP10; Index primer: HP12

sample Primer1 (P7) primer2 (S5) conc. (ng/ul) total (ng) vol (ul)
1 CAGOn/Cas- N701 S501 16.9 59 3.5
2 CAGOn/Cas+ N701 S502 5.9 59 10
3 CAG-OTS1/Cas- N701 S503 11.7 59 5
4 CAG-OTS1/Cas+ N701 S504 14.5 59 4.1
5 CAG-OTS2/Cas- N701 S505 11.2 59 5.3
6 CAG-OTS2/Cas+ N701 S506 15.9 59 3.7
7 CAG-OTS3/Cas- N701 S507 20.6 59 2.9
8 CAG-OTS3/Cas+ N701 S508 13.6 59 4.3
9 CAG-OTS4/Cas- N702 S501 21 59 2.8
10 CAG-OTS4/Cas+ N702 S502 17.6 59 3.4
11 CAG-OTS5Cas- N702 S503 16.8 59 3.5
12 CAG-OTS5Cas+ N702 S504 15.2 59 3.9
13 CAG-OTS6/7Cas- N702 S505 23.2 59 2.5
14 CAG-OTS6/7Cas- N702 S506 19.6 59 3
15 CAG-OTS8Cas- N702 S507 20.8 59 2.8
16 CAG-OTS8Cas+ N702 S508 22.8 59 2.6
17 CAG-OTS9Cas- N703 S501 17.6 59 3.4
18 CAG-OTS9Cas+ N703 S502 19 59 3.1
19 CAG-OTS10Cas- N703 S503 17.4 59 3.4
20 CAG-OTS10Cas+ N703 S504 21.6 59 2.7
21 CAG-OTS11Cas- N703 S505 17 59 3.5
22 CAG-OTS11Cas+ N703 S506 16.9 59 3.5
23 CAG-OTS12Cas- N703 S507 17.2 59 3.4
24 CAG-OTS12Cas+ N703 S508 23.1 59 2.6
  • data analysis
 bwa mem -O 4 -R '@RG    ID:      SM:OTS PL:ILLUMINA     LB:' /home/zhl002/genomeDB/mm9/Mm9.fasta $fastq->$sam
/home/kunzhang/softwares/samtools-latest/samtools view -b -t /home/zhl002/genomeDB/mm9/Mm9.fasta.fai $sam -> $bam
java -Xmx4g -jar /home/shg047/software/picard-tools-1.113/SortSam.jar TMP_DIR=./tmp/ INPUT=$bam OUTPUT=$.sorted.bam QUIET=True
SORT_ORDER=coordinate VALIDATION_STRINGENCY=SILENT
/home/kunzhang/softwares/samtools-latest/samtools index $.sorted.bam
/home/zhl002/software/freebayes/bin/freebayes -f /home/zhl002/genomeDB/mm9/Mm9.fasta -F 0.0001 -C 2 --pooled-continuous --no-snps --targets OTS$.bed 
$.sorted.bam
(for on-target bam file): -F 0.01 was used instead
GATK parameter used:
java -Djava.io.tmpdir=./tmp -Xmx4g -jar /home/kunzhang/softwares/GenomeAnalysisTK-3.3/GenomeAnalysisTK.jar -T UnifiedGenotyper -L chr18 
-R /home/zhl002/genomeDB/mm9/Mm9.fasta -I $.sorted.bam -glm INDEL -stand_call_conf 30 -stand_emit_conf 10 -o $.vcf
Calculate the in/del frequency: 
find alternative alleles identified at or close to the nuclease cutting site
take the column with insertion/deletion/complex alternative allele being called (AO), then divide the total read count in the vcf generated (DP) 
NOTES:
On target samples were mapped to custom sequences that span the targeting sites
Dr. Zhang repeated the analysis using script below:
/home/kunzhang/softwares/bwa-0.7.10/bwa bwasw  Ai14.fa CAGOnS1.fastq > CAGOnS1.bwasw.sam
samtools import Ai14.fa.fai CAGOnS1.bwasw.sam CAGOnS1.bwasw.bam
samtools sort CAGOnS1.bwasw.bam CAGOnS1.bwasw.sorted
samtools index CAGOnS1.bwasw.sorted.bam 
/home/kunzhang/softwares/freebayes/bin/freebayes -f Ai14.fa -F 0.0001 -C 2 --pooled-continuous  --target Ontarget.bed --no-snps CAGOnS1.bwasw.sorted.bam >  CAGOnS1.bwasw.sorted.vcf

Results[edit]

# chromosome position sequence PAM total read depth # of indel (%)
On chr6 GAGGAACTTCTTAGGGCCCGCGG CGG 717,296 4.2618
OTS1 chr13 9534141 CAGGAACTTCTTAGGTCCCGTGG TGG 825,966 0.0115
OTS2 chr14 85157555 CGGAAACATCTTAGGGCCCGGGG GGG 455,049 0.0147
OTS3 chr2 74925639 GGAGTACTTCTTAGGGCCCACAG CAG 407,292 0.0152
OTS4 chr15 69159526 CAGGAACATATTAGGGCCCAGGG GGG 364,310 0.0159
OTS5 chr12 100704049 CAGGAACATGTTAGGGTCCGAGG AGG 980,881 0.0114
OTS6 chrX 91969928 CAGGAACGTGTTCGGGCCCGCGG CGG 551,434 0
OTS7 chrX 92043584 CAGGAACGTGTTCGGGCCCGCGG CGG 551,434 0
OTS8 chr10 17440682 CAGGAACTTCTTAGTGCCCTAAG AAG 524,379 0.0147
OTS9 chr18 25702486 GTGGACCTTTTCAGGGCCCGTGG TGG 575,965 0.0325
OTS10 chr18 9212447 GAGGCCCCTCTTCGGGCCCGGAG GAG 484,996 0.0115
OTS11 chr14 46862821 GTGGAGCATCTTAGGGCCAGTGG TGG 745,785 0.0126
OTS12 chr12 31652981 GAGCAACAACTTAGGGCCTGCAG CAG 352,693 0.0139

OLD results of first few rounds of pilot experiment[edit]

  • 1st round primer design:
  • post-Nextera 1st PCR:
  • Kei-ValidSeq-F: ACATGGTCCTGCTGGAGTTC (Forward primer at end of GFP to detect the on/off target sequence after the knock-in site)
  • kei-ValidSeq-R: GTTTACGTCGCCGTCCAG (Reverse primer at beginning of GFP to detect the on/off target sequence before the knock-in site)
  • 2nd round primer design:
  • NOTE: previous design wasn't good because Kei's sample relied on NHEJ to integrate transgene while H9CFP used HR; the integration site wasn't exactly next to the fluorescent proteins, NHEJ sample is next to the cutting site while HR sample is next to HR arms
  • Kei-begGFP-R: GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGCTGAACTTGTGGCCGTTTA (Reverse primer at beginning of GFP to detect the on/off target sequence before the knock-in site)
  • Nextera library construction on extracted gDNA of FACS sorted GFP-positive cells:
  • TD (Tagment DNA Buffer)
  • NT (Neutralize Tagment Buffer)
  • ATM (Amplicon Tagment Mix)
  • Input DNA (protocol recommend 0.2 ng/μl)
Sample:
CRISPR mediated knock-in at Rosa26 transgenic mice(mCherry+) into non-dividing cells (injected into muscle cells)
Kei sample 3 (~4500 cells): -Cas9 mCherry+ gDNA
Kei sample 4 (~1500 cells): +Cas9 mCherry+ gDNA
Kei sample 6 (~320 cells): +Cas9 GFP+ gDNA
CFP-H9-BB (~7x10^5 cells) extracted DNA with Arcturus PicoPure DNA Extraction Kit provided by Kei (this kit can potentially used for very low cell#)

Tagmentation (Nextera XT Tagment Amplicon tube)

  1. Remove the ATM, TD, and input DNA from -25°C to -15°C storage and thaw on ice
  2. Visually inspect NT to make sure that there is no precipitate. If there is precipitate, vortex until all particulates are resuspended
  3. After thawing, mix reagents by gently inverting the tubes 3–5 times, followed by a brief spin in a microcentrifuge
  4. Add 2.5 μl TD Buffer to each well to be used in this assay
  5. Add 1.25 μl input DNA at 0.2 ng/μl (1 ng total)
  6. Add 1.25 μl ATM to the wells containing input DNA and TD Buffer.
  7. Centrifuge at 280 × g at 20°C for 1 minute.
  8. Place in a thermal cycler and run the following program: 55°C for 5 minutes -> Hold at 10°C
  9. When the sample reaches 10°C, proceed immediately to Neutralize samples as the transposome is still active and add 1.25 μl NT Buffer
  10. vortex at medium speed briefly
  11. Centrifuge at 280 × g at 20°C for 1 minute.
  12. Leave at room temperature for 5 minutes.

PCR amplification of tagmentation treated DNA

  1. add 1.25 μl index 2 primers (white caps)
  2. add 1.25 μl index 1 primers (orange caps)
  3. add 3.75 ul NPM (amplification mix)
  4. Centrifuge at 280 × g at 20°C for 1 minute.
  5. Perform PCR using the following program on a thermal cycler:
  • 72°C for 3 minutes
  • 95°C for 30 seconds
  • 12 cycles of: 95°C for 10 seconds, 55°C for 30 seconds, 72°C for 30 seconds
  • 72°C for 5 minutes
  • Hold at 10°C
  • Ampure bead XP purification with 1x concentration of bead, and elute in 10ul of H2O

2nd PCR amplification with custom primers

reaction setup (x4 samples):
purified gDNA from above: 3ul
Phusion MM:  25ul
Syb_FP5 (forward primer, 10uM):  1ul
kei-ValidSeq-R (reverse primer, 10um):  1ul
H2O:  20ul
2nd reaction setup (x4 samples): 
purified gDNA from above: 3ul
Phusion MM:  25ul
Syb_RP7(reverse primer, 10uM):  1ul
kei-ValidSeq-F (forward primer, 10um):  1ul
H2O:  20ul
  • 98°C for 30 seconds --> 28-35 cycles of: 98°C for 10 seconds, 60°C for 30 seconds, 72°C for 30 seconds --> 72°C for 3 minutes --> Hold at 10°C
  • gel results:

File:ZhangLab 2 2015-10-26 16hr 13min-edit.jpg

    • lane legend (left to right):
    • LM ladder
    • H9CFP-BB beginning of GFP
    • Kei: +Cas9+mCherry beginning of GFP
    • Kei: +Cas9+GFP beginning of GFP (30 cycles)
    • Kei: -Cas9+mCherry beginning of GFP
    • Kei: +Cas9+mCherry end of GFP
    • Kei: +Cas9+GFP end of GFP (30 cycles)
    • Kei: -Cas9+mCherry end of GFP
    • Kei: +Cas9+GFP beginning of GFP (purify and ran 5 more cycles)
    • Kei: +Cas9+GFP end of GFP (purify and ran 5 more cycles)

testing out different design of the PCR

  • after nextera, use the fragmented gDNA as template to run locus specific PCR
    • Kei-begGFP-R: (GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG)GCTGAACTTGTGGCCGTTTA
    • (sequences) indicates illumina adaptor overhang
reaction setup (x3 samples): 
gDNA after nextera: 6.25 ul
Kapa Hifi Readymix:  12.5ul
custom reverse primer (10uM):  1ul
F5 forward primer from Nextera:  1ul
H2O:  4.25ul
  • 95°C for 3 min --> 25 cycles of: 95°C for 30 seconds, 55°C for 30 seconds, 72°C for 30 seconds --> 72°C for 3 minutes --> Hold at 10°C

File:ZhangLab 2 2015-10-30 10hr 12min-edit.jpg

    • lane legend (left to right)
    • LM ladder
    • Kei: +Cas9+mCherry beginning of GFP
    • Kei: +Cas9+GFP beginning of GFP
    • Kei: -Cas9+mCherry beginning of GFP
  • purify the above PCR product with qiagen qiaquick kit and also Ampure bead
  • then amplify again in 50ul reaction with the same condition as above
  • gel results:

File:ZhangLab 2 2015-10-30 12hr 46min-edit.jpg

  • LAM-PCR product library construction*
  • Since the method above don't seem to work well, so maybe directly perform PCR with Illumina tail on genomic DNA is not the best way to go
  • there could be multiple reasons that the above method didn't work: complex genome, PCR primer tail bring
  • used new trial kit: NEBNext® Ultra™ II DNA Library Prep
  • followed manufacture protocol
  • sequencd in Miseq (~0.2 million reads total)