title: GSM3901919: E15.5 microglia from CD1 embryos; Mus musculus; RNA-Seq
library_construction_protocol: E15.5 brains were dissected from one wild-type control CD1 dam (n = 10 brains, hypothalamic/thalamic tissue pooled) and one stressed CD1 dam (n = 5 brains, hypothalamic/thalamic tissue pooled) in cold Hank's Buffered Salt Solution (HBSS; Life Technologies) on ice, where the hypothalamic/thalamic region was micro-dissected out and then placed in our live-cell media (DMEM, F12, GlutaMAX, FBS, HS, N2, B27 mixture) and chopped into smaller pieces using a blade while on ice. The cell suspension was then run through 70 µm cell filter and centrifuged at 300 x g. for 5 min at 4 degrees. Liberated single cells were re-suspended in 500 µL  of our live-cell media, and stained with 3 µL of Cd11b-PE and 3 µL of Cd45-FITC for 45 min at 4 degrees on a shaker in the dark. The staining cell suspension was then washed in 10mL of HBSS + 5% FBS on ice, spun at 1200rpm ( ~300 x g in a table-top centriduge) for 5 min at 4 degrees. The cells were resuspended in 300 µL of HBSS + 5% FBS and filtered through a 70 µm cell filter. For FACS purification, a stringent initial forward and side scatter gates were used to exclude debris, and cell doublets. Cd11b-PE / Cd45-FITC double positive cells were purified (10-12 min total length for each run) and collected in 100% FBS. The cells were then spun at 300 x g for 5 min at 4 degrees to achieve a final volume of ~40-50 µL to be used for 10X. Libraries were prepared according to 10X Genomics ChromiumTM Single Cell 3' Reagent Guidelines v3 10X Gene expression solution (using Chip B) as per the manufacturer's protocol. In brief, single cells were FACS sorted into 100% FBS 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 D1000 ScreenTape System. Next Generation Sequencing was done using Illumina HiSeq 4000 PE (75 bp paired-end reads) with a targeted sequencing depth of ~100,000 reads/cell. We recovered 1,156 cells from E15.5 control tissue (pooled) and 1,467 cells from E15.5 stressed tissue (pooled), with an estimated doublet rate of ~3-4%.