Both scDNase-seq and scMNase-seq are developed from Keji Zhao's lab. They use almost the same procudures to construct the library. One uses DNase to get DNA from open chromatin and the other uses MNase to get DNA from both open chromatin and nulcesomal regions. The methods utilise the traditional way of making libraries, which is ligating the sequencing adaptors to the DNase/MNase fragmented DNA. The trick is to add circular plasmid as carrier DNA during ligation and purification to reduce DNA loss. Therefore, the library structure is just standard Illumina sequencing libraries. Each cells are prepared separately, so the library index is the cell barcode.
Illumina adaptor top: 5'- /Phos/
Illumina adaptor bottom: 5'-
Make Illumina adaptors by annealing the top and the bottom sequences:
5'- ACAC TCTTTCCCTACACGAC GCTCTTCCGATCT -3'CGAGAAGGCTAG -5' 3'-TCTGCACA
Illumina PCR Primer 1.0: 5'-
Illumina Multiplexing PCR Primer: 5'-
Illumina P5 adapter: 5'-
Illumina P7 adapter: 5'-
Illumina TruSeq Read 1 primer: 5'-
Illumina TruSeq Read 2 primer: 5'-
Sample index sequencing primer: 5'-
5'- XXXXXXXXXXXXXXXXX...XXXXXXXXXXXXXXXXXA -3' 3'- AXXXXXXXXXXXXXXXXX...XXXXXXXXXXXXXXXXX -5'
5'- ACAC TCTTTCCCTACACGAC ACACGTCT -3'GCTCTTCCGATCT XXXXXXXXXX...XXXXXXXXXXAGATCGGAAGAGC CGAGAAGGCTAG AXXXXXXXXXX...XXXXXXXXXXTCTAGCCTTCTCG 3'-TCTGCACA CAGCACATCCCTTTCT CACA -5'
(i) First round (the product from the top and bottoms strands have the same structure): Top strand: 5'- ACAC TCTTTCCCTACACGACGCTCTTCCGATCT XXXXXXXXXX...XXXXXXXXXXAGATCGGAAGAGCACACGTCT -3' <----------TCTAGCCTTCTCGTGTGCAGACTTGAGGTCAGTG [i7]TAGAGCATACGGCAGAAGACGAAC -5' Bottom strand: 5'-CAAGCAGAAGACGGCATACGAGAT [i7]GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT ------------> 3'-TCTGCACACGAGAAGGCTAG AXXXXXXXXXX...XXXXXXXXXXTCTAGCCTTCTCGCAGCACATCCCTTTCT CACA -5' (ii) Second round and after: 5'-AATGATACGGCGACCACCGAGATCTACAC TCTTTCCCTACACGACGCTCTTCCGATCT ------------> 5'-ACAC TCTTTCCCTACACGACGCTCTTCCGATCT XXXXXXXXXX...XXXXXXXXXXAGATCGGAAGAGCACACGTCTGAACTCCAGTCAC [i7]ATCTCGTATGCCGTCTTCTGCTTG -3' 3'-TGTG AGAAAGGGATGTGCTGCGAGAAGGCTAGA XXXXXXXXXX...XXXXXXXXXXTCTAGCCTTCTCGTGTGCAGACTTGAGGTCAGTG [i7]TAGAGCATACGGCAGAAGACGAAC -5' <----------TCTAGCCTTCTCGTGTGCAGACTTGAGGTCAGTG [i7]TAGAGCATACGGCAGAAGACGAAC -5'
5'- AATGATACGGCGACCACCGAGATCTACAC TCTTTCCCTACACGACGCTCTTCCGATCT XXX...XXXAGATCGGAAGAGCACACGTCTGAACTCCAGTCAC NNNNNNATCTCGTATGCCGTCTTCTGCTTG -3' 3'-TTACTATGCCGCTGGTGGCTCTAGATGTG AGAAAGGGATGTGCTGCGAGAAGGCTAGA XXX...XXXTCTAGCCTTCTCGTGTGCAGACTTGAGGTCAGTG NNNNNNTAGAGCATACGGCAGAAGACGAAC -5'Illumina P5 TruSeq Read 1 gDNATruSeq Read 2 6-bpIllumina P7 i7 index
5'- ACAC TCTTTCCCTACACGACGCTCTTCCGATCT ------> 3'-TTACTATGCCGCTGGTGGCTCTAGATGTG AGAAAGGGATGTGCTGCGAGAAGGCTAGA XXX...XXXTCTAGCCTTCTCGTGTGCAGACTTGAGGTCAGTG NNNNNNTAGAGCATACGGCAGAAGACGAAC -5'
5'- GATCGGAAGAGCACACGTCTGAACTCCAGTCAC -----> 3'-TTACTATGCCGCTGGTGGCTCTAGATGTG AGAAAGGGATGTGCTGCGAGAAGGCTAGA XXX...XXXTCTAGCCTTCTCGTGTGCAGACTTGAGGTCAGTG NNNNNNTAGAGCATACGGCAGAAGACGAAC -5'
5'- AATGATACGGCGACCACCGAGATCTACAC TCTTTCCCTACACGACGCTCTTCCGATCT XXX...XXXAGATCGGAAGAGCACACGTCTGAACTCCAGTCAC NNNNNNATCTCGTATGCCGTCTTCTGCTTG -3' <------TCTAGCCTTCTCGTGTGCAGACTTGAGGTCAGTG -5'