title: GSM8417405: 3xTg_42; Mus musculus; RNA-Seq
library_construction_protocol: Patch-seq required slight modifications to recording parameters to minimize confounds in both electrophysiological recordings and sequencing. Patch-seq internal was a modification of whole-cell internal solution containing (in mM) 124 K gluconate, 10 HEPES, 5 KCl, 5 MgCl2, 0.1 EGTA, 0.075 CaCl2, 20 µg/mL glycogen, and 0.5 units/µL RNase inhibitor (M0314L, New England Biolabs). The voltage step protocol used to measure the tail current was unchanged. To minimize recording time before cell extraction, additional electrophysiological parameters were not recorded. After recording was complete, light negative pressure was applied to the pipette until the nucleus was visible at the tip of the pipette, or for approximately three minutes. Next, the recording pipette was slowly retracted along the Z-axis. Successful extractions included an obvious nucleated patch67. After the nucleus was visualized above the slice, the pipette was rapidly retracted from the bath, and the cellular contents within the pipette were deposited in a 0.2 mL PCR tube containing 0.5 µL of NEB Next lysis buffer and 0.25 µL NEB murine RNase inhibitor. Tube contents were then rapidly spun down and flash frozen on dry ice. Samples were stored at -80°C until RNAseq libraries were generated. Directional libraries were constructed using NEBNext Single Cell/Low Input RNA Library Prep Kits from Illumina following manufacturer instructions. In brief, poly-adenylated mRNA was magnetically captured and then eluted from the oligo-dT beads. mRNA was then fragmented and reverse-transcribed, and the cDNA was purified using SPRISelect beads (#B23318, Beckman Coulter). Purified cDNA was eluted in 50 μL 0.1 × TE buffer. Adaptors were diluted and ligated to cDNA, and the cDNA was amplified by 28 cycles of PCR. SPRISelect beads were used to select for proper library size. Purified library quality was then assessed on a tapestation (#4200, Agilent Technologies) using HSD1000 screentapes (#5067–5584; Agilent Technologies). Due to the high number of amplification cycles, occasionally libraries required additional rounds of SPRISelect purification. Qubit 1 × dsDNA HS Assay kit (#Q33230, Thermo Fisher Scientific) was used to quantify libraries. Libraries were pooled and sequenced 2x 100 bases on an Illumina MiSeq. Fastq data will be made available on the gene expression omnibus (GEO) upon acceptance.