Instrument: Illumina HiSeq 2500
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: SINGLE
Construction protocol: Individual adult male mice (P56±3) were anesthetized in an isoflurane chamber, decapitated, and the brain was immediately removed and submerged in fresh ice-cold artificial cerebrospinal fluid (ACSF). The brain was sectioned on a vibratome (Leica VT1000S) on ice, and each slice (300-400 µm) was immediately transferred to an ACSF bath at room temperature. After the brain slicing is complete (not more than 15 minutes), individual slices of interest were transferred to a small Petri dish containing bubbled room temperature ACSF. The regions of interest (all layers of VISp or specific layers of VISp) were microdissected under a fluorescence dissecting microscope, and the slices before and after dissection were imaged to later examine the location of the microdissected tissue and confirm its location within V1. The dissected tissue pieces were transferred to a microcentrifuge tube and treated with 1 mg/ml pronase in carbogen-bubbled ACSF for 70 minutes at room temperature without mixing in a closed tube. After incubation, with the tissue pieces sitting at the bottom of the tube, the pronase solution was pipetted out of the tube and exchanged with cold ACSF containing 1% fetal bovine serum. The tissue pieces were dissociated into single cells by gentle trituration through Pasteur pipettes with polished tips of 600, 300, and 150-µm diameter. Single cells were isolated by FACS into individual wells of 96-well plates or 8-well PCR strips containing 2.275 µl of Dilution Buffer (SMARTer Ultra Low RNA Kit for Illumina Sequencing, Clontech Cat#634936), 0.125 µl RNase inhibitor (SMARTer kit), and 0.1 µl of 1:1,000,000 diluted RNA spike-in RNAs (ERCC RNA Spike-In Mix 1, Life Technologies Cat#4456740). Sorting was performed on a BD FACSAriaII SORP using a 130um nozzle, a sheath pressure of 10psi, and in the single cell sorting mode. To exclude dead cells, DAPI (DAPI*2HCl, Life Technologies Cat#D1306) was added to the single cell suspension to the final concentration of 2 ng/ml. FACS populations were chosen to select cells with low DAPI and high tdTomato (tdT) fluorescence. In some cases, we also selected cells that have low DAPI and low tdT fluorescence, in order to capture tdT-negative cells from a sample. To collect all cells in an unbiased manner, we selected all cells with low DAPI fluorescence, regardless of their tdT fluorescence level. Sorted cells were frozen immediately on dry ice and stored at -80°C. We used the SMARTer kit from Clontech (SMARTer Ultra Low RNA Kit for Illumina Sequencing, Clontech Cat#634936) to reverse transcribe single cell RNA, and amplify the cDNA for 19 PCR cycles. To stabilize the RNA after quickly thawing the cells on ice, we immediately added to each sample an additional 0.125 µl of RNase inhibitor mixed with SMART CDS Primer II A. All steps downstream were carried out according to the manufacturer’s instructions. Each amplification experiment included a set of controls: 10 pg cortex RNA (isolated from Rbp4-Cre;Ai14, P57 male) as positive control for amplification, ERCC-only control to demonstrate the absence of RNases throughout the sorting process, and water-only control, to control for specificity of amplification/absence of contamination. cDNA concentration was quantified using Agilent Bioanalyzer High Sensitivity DNA chips. For most samples, 1 ng of amplified cDNA was used as input to make sequencing libraries with Nextera XT DNA kit (Illumina Cat#FC-131-1096). For smaller cells (e.g., glia), which did not consistently produce more than 1ng cDNA, we used 0.5–1 ng cDNA as input. We stopped the procedure after PCR clean-up and did not perform library normalization or library pooling. Individual libraries were quantified using Agilent Bioanalyzer DNA 7500 chips. In order to assess sample quality and adjust the concentrations of libraries for multiplexing on HiSeq, all libraries were sequenced first on Illumina MiSeq to obtain approximately 100,000 reads per library, and then on Illumina HiSeq 2000 or 2500 to generate 100 bp reads.