DR-seq was published by Dey et al. in Nature Biotechnology 33, 285-289. It is a method to simultaneously analyse gDNA and mRNA without upfront physical separation, though it perform library part separately for gDNA and mRNA in the later part of the protocol. It uses MALBAC type of apmification to preamplify gDNA and cDNA together. Then, PCR is used to construct gDNA library and IVT is used to construct the mRNA library. There will be some mRNA reads in the gDNA portion of the library.
Barcoded RT primer: 5'- CGATTGAGGCCGG
T7 promoter: 5'-
2nd strand primer: 5'- CGATTGAGGCCGG
MALBAC random primers: 5'-
MALBAC PCR primer: 5'-
gDNA: 5'- XXXXXXXXXXXX...XXXXXXXXXXXX -3' 3'- XXXXXXXXXXXX...XXXXXXXXXXXX -5' mRNA: 5'- XXXXXXXXXXXX...XXXXXXXXXXXXB(pA) -3' <-----------V(dT) [8-bp cell barcode] CTAGCAGCCTGACATCTTGAGACTTG GGGATATCACTCAGCATAATGGCCGGAGTTAGC -5'
gDNA Procduct 1 (not amplifiable by PCR in the next step): 5'- GTGAGTGATGGTTGAGGTAGTGTGGAGXXXXXXXXXXXX...XXXXXXXXXXXX -3' gDNA Product 2 (amplifiable by PCR in the next step): 5'- GTGAGTGATGGTTGAGGTAGTGTGGAGXXXXXX...XXXXXX CTCCACACTACCTCAACCATCACTCAC-3' mRNA Product 1 (not ampliable by PCR or IVT in the next step): 5'- GTGAGTGATGGTTGAGGTAGTGTGGAGXXXXXXXXXXXX...XXXXXXXXXXXX -3' mRNA Product 2 (ampliable by PCR but not IVT in the next step, this portion will be mistakenly as gDNA): 5'- GTGAGTGATGGTTGAGGTAGTGTGGAGXXXXXX...XXXXXX CTCCACACTACCTCAACCATCACTCAC-3' mRNA Product 3 (not ampliable by PCR but amplifiable by IVT in the next step): 5'- GTGAGTGATGGTTGAGGTAGTGTGGAGXXXXXX...XXXXXXB(pA) [8-bp cell barcode] GATCGTCGGACTGTAGAACTCTGAAC CCCTATAGTGAGTCGTATTACCGGCCTCAATCG -3'
One half is used to amplify gDNA using PCR with MALBAC PCR primer. NOTE mRNA Product 2 from above will also be amplified: 5'- GTGAGTGATGGTTGAGGTAGTGTGGAG------> 5'- GTGAGTGATGGTTGAGGTAGTGTGGAGXXXXXXXXXXXXX...XXXXXXXXXXXXX CTCCACACTACCTCAACCATCACTCAC-3' <------ GAGGTGTGATGGAGTTGGTAGTGAGTG-5' The other half is used to amplify cDNA using IVT: First, 2nd strand synthesis is performed using 2nd strand primer, which aims to capture mRNA Product 3 from above: 5'- GTGAGTGATGGTTGAGGTAGTGTGGAGXXXXXX...XXXXXXB(pA) [8-bp cell barcode] GATCGTCGGACTGTAGAACTCTGAAC CCCTATAGTGAGTCGTATTACCGGCCTCAATCG -3' <---------- CATAATGGCCGGAGTTAGC -5' Then, use IVT to amplify the double stranded cDNA: 5'- GTGAGTGATGGTTGAGGTAGTGTGGAGXXXXXX...XXXXXXB(pA) [8-bp cell barcode] GATCGTCGGACTGTAGAACTCTGAAC CCCTATAGTGAGTCGTATTACCGGCCTCAATCG -3' 3'- CACTCACTACCAACTCCATCACACCTCXXXXXX...XXXXXXB(dT) [8-bp cell barcode] CTAGCAGCCTGACATCTTGAGACTTG GGGATATCACTCAGCATAATGGCCGGAGTTAGC -5' ↵ IVT starts from here
Uisng Nextera: 5'- AATGATACGGCGACCACCGAGATCTACACNNNNNNNN TCGTCGGCAGCGTC AGATGTGTATAAGAGACAGXXXXXXXX...XXXXXXXX CTGTCTCTTATACACATCT CCGAGCCCACGAGACNNNNNNNN ATCTCGTATGCCGTCTTCTGCTTG TTACTATGCCGCTGGTGGCTCTAGATGTGNNNNNNNN AGCAGCCGTCGCAG TCTACACATATTCTCTGTCXXXXXXXX...XXXXXXXX GACAGAGAATATGTGTAGA GGCTCGGGTGCTCTGNNNNNNNN TAGAGCATACGGCAGAAGACGAAC-5' Illumina P5i5 s5 MEgDNA ME s7i7 Illumina P7Uisng TruSeq: 5'- AATGATACGGCGACCACCGAGATCTACAC TCTTTCCCTACACGACGCTCTTCCGATCTXXX...XXX AGATCGGAAGAGCACACGTCTGAACTCCAGTCACNNNNNNNN ATCTCGTATGCCGTCTTCTGCTTG-3' 3'- TTACTATGCCGCTGGTGGCTCTAGATGTG AGAAAGGGATGTGCTGCGAGAAGGCTAGAXXX...XXX TCTAGCCTTCTCGTGTGCAGACTTGAGGTCAGTGNNNNNNNN TAGAGCATACGGCAGAAGACGAAC-5' Illumina P5 Truseq Read 1gDNA Truseq Read 28 bp Illumina P7Index
5'- AATGATACGGCGACCACCGAGATCTACAC GTTCAGAGTTCTACAGTCCGACGATC NNNNNNNN(dT)XXX...XXX TGGAATTCTCGGGTGCCAAGGAACTCCAGTCACNNNNNN ATCTCGTATGCCGTCTTCTGCTTG-3' 3'- TTACTATGCCGCTGGTGGCTCTAGATGTG CAAGTCTCAAGATGTCAGGCTGCTAG NNNNNNNN(pA)XXX...XXX ACCTTAAGAGCCCACGGTTCCTTGAGGTCAGTGNNNNNN TAGAGCATACGGCAGAAGACGAAC-5' Illumina P5 RA5 8bpcDNA RA36bp Illumina P7 cellsample barcodebarcode
Standard sequencing workflow is used, and you can check any other pages.