GSM4060293: Spata7_WT_3M; Mus musculus; RNA-Seq
Using ICELL8 multisample nanodispenser (Wafergen Biosystems), 50nl (3E4/ml) of the live cell or nuclear suspension was precisely dispensed into an ICELL8 nanowell microchip containing 5184 wells (150nl capacity) as described previously (Goldstein et al., 2017). Assuming a Poisson distribution frequency for the number of cells per well, about 30% of the nanowells were expected to contain a single nucleus under optimal conditions. Automated scanning fluorescent microscopy (using Olympus BX43 fluorescent microscope with a robotic stage) of the microchip was used to select ~1400 wells containing a single live cell or a single nucleus (CellSelect software, Wafergen Biosystems).  The candidate wells were manually evaluated for debris or clumps as an additional QC. The chip was subjected to freeze-thaw in order to lyse the cells and 50 nl of reverse transcription and amplification solution (following ICELL8 protocol) was dispensed using the multisample nanodispenser to candidate wells. SCRB-seq was then performed on the candidate wells using pre-printed primer that adds a well-specific barcode to the (A)n mRNA tail of the cellular transcript. The cDNA libraries were pooled, concentrated (Zymo Clean and Concentrator kit, Zymogen) and purified (using 0.6x AMPure XP beads). 3' transcriptome enriched library was made by Nextera XT Kit and 3'-specific P5 primer, and sequenced on Illumina Hiseq2500.
GEO Accession
GSM4060293