GSM3172589: E15.5 Microglia Cx3cr1Cre:tdTomato+ve; Mus musculus; RNA-Seq
E15.5 brains were dissected from one dam (n = 5 brains pooled) and enzymatically dissociated with Accutase (StemCell Technologies) for 15 mins at 37oC. Cell suspensions were diluted with cold Hank's Buffered Salt Solution (HBSS; Life Technologies), strained through 70 µm cell filters and centrifuged at 259 x g. Liberated single cells were re-suspended in 1% bovine serum album in HBSS before FACS purification. For FACS purification, a stringent initial forward and side scatter gates were used to exclude debris, and cell doublets. Cells from Cx3cr1CreERT2:tdTomato mice were purified. Libraries were prepared according to 10X Genomics ChromiumTM Single Cell 3' Reagent Guidelines v2 Chemistry as per the manufacturer's protocol. In brief, single cells were sorted into 0.1% BSA–PBS and partitioned into Gel Bead-In-EMulsions (GEMs) using 10xTM GemCodeTM Technology. This process lysed cells and enabled barcoded reverse transcription of RNA, generating full-length cDNA from poly-adenylated mRNA. DynaBeads® MyOneTM Silane magnetic beads were used to remove leftover biochemical reagents, then cDNA was amplified by PCR over 10 cycles. Quality control size gating was used to select cDNA amplicon size prior to library construction. Read 1 primer sequences were added to cDNA during GEM incubation. P5 primers, P7 primers, i7 sample index, and Read 2 primer sequences were added during library construction. Quality control and cDNA quantification was performed using Agilent High Sensitivity DNA Kit. Sequencing was performed first using Illumina MiSeq SR50 to approximate the number of recovered cells in each sample. We recovered 4061, 3776, 2604, and 3293 cells for Samples 1-4, respectively, with an estimated doublet rate of ≈3%. Based on this, we determined lane distributions for sequencing using Illumina HiSeq 4000 PE (75 bp paired-end reads) with a targeted sequencing depth of ~115,000 reads/cell.
GEO Accession
GSM3172589