GSM2414404: L5Cal-4; Mus musculus; RNA-Seq
Mice aged P35 or older were anaesthetized with ketamine and xylazine (100mg*kg-1 and 10mg*kg-1, respectively), perfused transcardially with cold sucrose solution (in mM: NaCl, 83; KCl, 2.5; MgSO4, 3.3; NaH2PO4, 1; NaHCO3, 26.2; D-glucose, 22; sucrose, 72; and CaCl2, 0.5, bubbled with 95% O2 and 5% CO2) and decapitated, and the visual cortex was cut into 300µm coronal or tangential sections in cold sucrose solution. Slices were incubated in sucrose solution in a submerged chamber at 34°C for 30min and then at room temperature (21°C) until used for RNA harvest from patch-clamped cells. RNA was harvested as described previously (Pfeffer et al., 2013). In brief, slices were mounted under a 40x 0.8 NA water lens and superfused with sucrose solution (same as in slice preparation). Cells were identified using GFP or tdTomato  fluorescence and visualized with IR-DIC. Cells were then patched with regular RNAse free patch pipettes (2-4 MOhm resistance) in whole-cell mode using the voltage-clamp configuration. After seal establishment and break-in the cell was held at -70mV and the cell content including cell nucleus was harvested using negative pressure and expelled in an Eppendorf tube and frozen at -70C. Intracellular solution was RNAse free: in mM: CsMeSO4, 115; NaCl, 4; HEPES, 10; Na3GTP, 0.3; MgATP, 4; EGTA, 0.3; BAPTA(4Cs), 10; adjusted to pH 7.4 with CsOH; mOsm 295. The procedures lasted approximately 2-3 minutes per cell (from targeting to tube freezing). Single cell RNA-seq was carried out following the Cel-seq protocol (Hashimshony et al., 2016; Hashimshony et al., 2012). RT and second strand synthesis were performed using Superscript III (Invitrogen) and second strand synthesis kit (Invitrogen), respectively, before IVT amplification (Maxiscript T7, Ambion). 96 samples (up to 94 cells and 2-8 control samples) were handled in parallel to construct one sequencing library. After library fragmentation (Mg-Acetat + heat) and 3’ adaptor attachment, 11-15 cycles of PCR were used for library amplification (final concentration > 1ng/microL, library peak fragment size ~350 nt). Libraries were sequenced using Illumina HiSeq 2500 and HiSeq 4000 at the UCSD genomics core (1st read 13 cycles for the barcode, 2nd read 50 cycles for the 3’ end of the transcript).
GEO Accession
GSM2414404