Instrument: Illumina HiSeq 2500
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: PAIRED
Construction protocol: Dentate gyrus was microdissected and cell populations were isolated into single cells using a Becton Dickinson FACS. Immediately after FACS, collected cells were centrifuged and then resuspended in cell culture medium at a concentration of 500 cells/μl. The cell suspension was then mixed with C1 Cell Suspension Reagent (Fluidigm, 634833) at the recommended ratio of 3:2 immediately before loading 5 μl of this final mix on the C1 Integrated Fluidic Circuit (IFC). Single cells were capture and RNA extraction was performed by the Fluidigm C1 IFC. mRNA amplification was performed on the C1 Single-Cell Auto Prep Integrated Fluidic Circuit (IFC) following the methods described in the protocol (PN 100-7168). The cDNA products were quantified using the Quant-iT PicoGreen double-stranded DNA (dsDNA) Assay Kit (Life Technologies) and high-sensitivity (HS) DNA chips (Agilent). All samples assayed had material present in the 1000-3000bp range with concentrations varied from ~160pg/μl to 1.5ng/µl. The cDNA samples were diluted to less than 0.3ng/μl and 2ul of diluted cDNA reaction prod¬ucts were then converted into libraries using the Nextera XT DNA Sample Preparation Kit (Illumina, FC-131-1096 and FC-131-1002) following the manufacturer’s instruction. Briefly, 12 Cycles of PCR Amplification were used. After the PCR step, samples were pooled, cleaned with 0.9× Agencourt AMPure XP SPRI beads (Beckman Coulter lot#14669400 X2), eluted in Tris + EDTA buffer and quantified using a HS DNA chip (Agilent).