GSM4874898: sc_Paired_tag_Brain_H3K4me3_sub_library_DNA#05; Mus musculus; OTHER
Libraries were constructed by Paired-tag workflow. First, permeabilized nuclei are incubated with antibodies targeting specific histone modifications (or other DNA binding proteins) to target the binding of protein A-fused Tn5 to chromatin. Tagmentation reaction and reverse transcription (RT) are then sequentially performed. Specifically, reactions are carried out in 12 different wells, each with a well-specific DNA barcode included in the transposase adaptors and RT primers, to label different samples or replicates (1st round of barcodes). Next, a ligation-based combinatorial barcoding strategy is used to introduce the 2nd and 3rd rounds of DNA barcodes to the nuclei, by sequentially attaching well-specific DNA barcodes to the 5'-end of both chromatin DNA fragments and cDNA from RT in 96-well plates. Finally, the barcoded nuclei are divided into sub-libraries and lysed, the chromatin DNA and cDNA are purified and used to prepare for sequencing libraries with an improved “amplify-and-split” strategy. For the RNA part, Tn5 transposases bound to the 2nd sequencing adaptor are used. For the DNA part, the fragment sizes of DNA from targeted tagmentation are shorter than those of cDNA from RT, which would result in lower library yields if Tn5 tagmentation is used to add the 2nd adaptor. Thus, to maximize efficiency, we used a type IIS endonuclease to create a nick and the 2nd sequencing adaptor is then introduced by ligation.
GEO Accession
GSM4874898