GSM5608373: nonVIP.100nuc.techrep3; Mus musculus; RNA-Seq
Freshly dissected visual cortices were snap frozen in liquid nitrogen and stored at -80C. Nuclei were then extracted,  stained with antibodies and Dapi and FACS-sorted  into 0.2ml low-bind tubes containing 2.03 µl lysis buffer. Libraries were prepared based on the Smart-Seq2 protocol with small modifications. Briefly, 100 nuclei were collected directly into 2.03 microliter lysis buffer (0.2% Triton X-100 in ddW) supplemented with dNTPs (2mM each), SMART-dT30VN primers (1mM), ERCC (1:1.25x107), and RNAse inhibitor. Reverse transcription was performed directly on isolated nuclei, followed by PCR-amplification (17 cycles). After cleanup with Agencourt Ampure XP beads (Beckman Coulter), the amplified cDNA underwent library generation using Nextera XT (Illumina). Libraries were quantified by Qubit (Thermo Fisher Scientific) and TapeStation (Agilent).