title: Two adjacent 50 microns thick hippocampus slices were sequenced - this one with RNAseq
design: See details in https://doi.org/10.1101/2020.05.13.094268. To perform a comparison of untargeted ExSeq to bulk RNAseq of a matching tissue sample, a coronal hippocampal slice was selected that was within 100 m of the original 50 micron thick hippocampal slice along the antero-posterior axis. The coronal hippocampal slice was fixed with 4% PFA in exactly the same way as the original slice, washed with 1X PBS and was kept in 70% ethanol at 4 until RNA extraction; throughout the experimental procedure of sequencing the RNA from a matching sample, we followed the steps of the ExSeq sequenced sample when possible. Accordingly, the DNA was removed using DNase I (Roche). RNA was extracted from these slices using an RNeasy FFPE Kit (Qiagen) following the Deparaffinization using Melting protocol indicated in the kit. The extracted RNA was then reverse transcribed using SSIV reverse transcriptase with random primers, following the manufacturer's protocol. The RNA was removed using RNase DNase-free (Roche) and RNase H. The single stranded cDNA was transformed into ds-cDNA with NEBNext second strand synthesis (New England Biolabs). After purification with the Genomic DNA Clean & Concentrator Kit (Zymo), 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 in vitro sequencing. The Nextera XT Index Kit (Illumina) was used to add Illumina sequencing barcodes during the PCR amplification of the extracted DNA. The library was sequenced using a NextSeq instrument was used to generate paired-end sequencing reads from the extracted DNA with NSQ 500 Mid Output Kit v2 (Illumina).