GSM4669508: mESC-scSPRITE-50%-01; Mus musculus; OTHER
mESC nuclei were isolated and permeabilized. In-nuclei restriction digest occurred followed by in-nuclei dA-tailing. 3 rounds of in-nuclei barcoding were performed to uniquely label DNA complexes inside individual nuclei with a cell-specific barcode. Sonication was done to lyse nuclei, and 3 round of DNA-complex barcoding was done to preserve spatial DNA information. After barcoding, DNA complexes were reverse crosslinked and DNA was purified for PCR. Crosslinked (with 1% formaldehyde, 2mM disuccinimidyl glutarate) chromatin was first digested in-nuclei with HpyCH4V restriction enzyme. DNA complexes in-nuclei were then ligated with a cell-specific DNA barcode by split-and-pooling over a 96 well-plate (where each well contains a unique DNA barcode) over 3 rounds. Nuclei are then sonicated to release DNA complexes, which still contain the cell-specific DNA barcodes. Three additional rounds of split-pool barcoding were done to add a spatial barcode, which identifies all DNA molecules in that DNA complex. Here, a total of 6 barcodes are added. DNA complexes are then reverse crosslinked, DNA is purified and amplified using Illumina-standard primers. Library is cleaned up by running on a 1% agarose gel, cutting the library between 300-1000 bp, and purifying the library using Zymo's gel clean-up columns. The library is then sequenced with paired-end sequencing.
GEO Accession
GSM4669508