A 15 microns thick hippocampus slice was sequenced with ExSeq and then the rolonies sequenced ex situ
See details in https://doi.org/10.1101/2020.05.13.094268. The specimen was first stripped for 30 min to remove the sequencing primer with strip solution, and washed with 1X instrument buffer for 10 min. The hydrogel was then digested with 20 mM sodium meta-periodate in 1X PBS pH 6 at 37C for 12 hours, followed by 2 min of vortexing. The DNA was extracted and purified from the digested hydrogel with Genomic DNA Clean & Concentrator Kit (Zymo Research), and then the single stranded DNA was transformed into dsDNA with NEBNext (New England Biolabs). After additional purification with the Genomic DNA Clean & Concentrator Kit, the Nextera XT DNA Library Preparation Kit (Illumina) was used to simultaneously fragment and tag the dsDNA with the adapter sequences required for the Illumina MiSeq in vitro sequencing. The Nextera XT Index Kit (Illumina) was used to add Illumina sequencing barcodes during the PCR amplification of the extracted DNA. For the 15 micron thick hippocampal slice a MiSeq instrument was used to generate paired-end sequencing reads from the extracted DNA with MiSeq Reagent Kit v3 ( Illumina).