单细胞全转录组扩增总结.docVIP

多重退火环状循环扩增技术MALBAC MALBAC single cell whole genome amplification. A single cell is picked and lysed. First, genomic DNA of the single cell is melted into single-stranded DNA molecules at 94°C. MALBAC primers then anneal randomly to single-stranded DNA molecules at 0°C and are extended by a polymerase with displacement activity at elevated temperatures, creating semi-amplicons. In the following five temperature cycles, after the step of looping the full amplicons, single stranded amplicons and the genomic DNA are used as template to produce full amplicons and additional semi-amplicons, respectively. For full amplicons, the 3′ end is complementary to the sequence on the 5′ end. The two ends hybridize will form the looped DNA, which can efficiently prevents the full amplicon from being used as template, therefore warrant a close-to-linear amplification. After the five cycles of linear preamplification, only the full amplicons can be exponentially amplified in the following PCR using the common 27-nucleotide sequence as the primer. PCR reaction will generate microgram level of DNA material for sequencing experiments. MALBAC覆盖率高、扩增均一，目前MALBAC试剂盒已经做到单管操作，经细胞裂解、MALBAC预扩增、指数式扩增三步，4小时内获得约2-4μg产物 SMART-seq2 TSO (5′-AAGCAGTGGTATCAACGCAGAGTACATrGrG+G-3′) Oligo-dT30VN (5′–AAGCAGTGGTATCAACGCAGAGTACT30VN-3′) ISPCR oligo (5′-AAGCAGTGGTATCAACGCAGAGT-3′) MDA ? MDA的扩增效率比MALBAC方法高，但各个片段的扩增倍数一致性则没有MALBAC方法好。?????相比之下，MALBAC方法测以扩增倍数的一致性见长，但扩增效率略低。 STRT Single-cell tagged reverse transcription sequencing (STRT-Seq) is a method similar to CEL-seq that involves unique barcoding and sample pooling to overcome the challenges of samples with limited material48. In this method, single cells are first picked in individual tubes, where firststrand cDNA synthesis occurs using an oligo(dT) primer with the addition of 3–6 cytosines. A helper oligo promotes template switching, which introduces the barcode on the cDNA. Barcoded cDNA is then amplified by single-primer PCR. Deep sequencing allows for