GSM5241149: L2-gDNA_S71; Homo sapiens; OTHER
Bulk forebrains were dissected from postnatal day (P)7 mice injected with the viral library at P0. Dissection included whole forebrain, separated into left and right hemispheres, after removing surface tissue and skull. Hemispheres were stored in RNAlater Stabilization Solution (Thermo Fisher #AM7020) for downstream dual DNA and RNA collection. Hemispheres were then physically homogenized and processed using the AllPrep DNA/RNA Mini Kit (Qiagen #80204) to collect total RNA and genomic DNA from the same tissue. RNA was treated on-column with 1 µL RNase-Free DNase (Qiagen #79254). First strand cDNA was synthesized from purified total RNA using the SuperScript VILO cDNA Synthesis Kit (Invitrogen #11755050) with random primers. We amplified the variable regions of the reporter library from cDNA and genomic (g)DNA using PCR with Phusion Hot Start II High Fidelity DNA polymerase (Invitrogen #F549S) using primers flanking the variable region within pscAAV-Hsp68-EGFP (forward primer, Amplicon PCR Primer F: GATCACTCTCGGCATGGAC; reverse primer, Amplicon PCR Primer R: GATGGCTGGCAACTAGAAGG). PCR consisted of 30 cycles (or 35, for technical replicate Sample 4-35) at 60 °C for annealing and 1 min for extension. PCRs were then loaded onto a 1.0% agarose gel, the ~1-1.1 kb fragment excised, and gel fragment purified using the Promega Gel & PCR Cleanup system (#A9282). Purified gel fragments were then re-purified using the Qiagen MinElute PCR Purification Kit (#28004) to remove residual chemical contamination, and concentration and quality assessed with Nanodrop and the Qubit dsDNA High Sensitivity assay kit (Thermo Fisher #Q33230). Purified amplicons were prepared for sequencing using the Nextera XT DNA Library Preparation Kit (Illumina #FC-131-1001) using 1 ng of purified amplicon library per sample. Quality and concentration of sequencing libraries was assessed using an Agilent BioAnalyzer instrument. Each library was quantified and pooled for sequencing on the Illumina HiSeq 4000 using a paired end 150-bp sequencing strategy. The miniMPRA was sequenced on a MiSeq instrument since lower coverage was required due to lower library complexity.