title: GSM9149591: GEM-X Flex Gene Expression, pool of 4 samples (Pool 1); Mus musculus; RNA-Seq
library_construction_protocol: Nuclei were isolated from frozen mouse hemi brains using Dounce homogenization (10 strokes of Pestle A, 20 strokes of Pestle B) in 1 mL of cold homogenization buffer (250 mM sucrose, 25 mM KCl, 5 mM MgCl2, 20 mM Tricine-KOH, 1 mM DTT, 0.5 mM spermidine, 0.15 mM spermine, 1% Triton) containing RNase inhibitor in a 2 mL Dounce, followed by preparation of an Iodixanol gradient (25%, 30%, 40% Iodixanol layers) and density gradient centrifugation in a swinging bucket centrifuge. Nuclei appeared as a band between the 30% and 40% Iodixanol layers after centrifugation. Nuclei were washed, counted, and centrifuged at 350 rcf. After removing the supernatant the nuclei pellet was resuspended in Fixation Buffer B containing 4% Formaldehyde from the 10x Genomics GEM-X Flex Sample Preparation Kit v2. Nuclei samples were fixed overnight at 4 degrees C before addition of Additive C reagent and being quenched with Quenching Buffer B provided with the Sample Preparation Kit. GEM-X Flex Gene Expression libraries were prepared using the 10x Genomics User Guide CG000787 (GEM-X Flex Reagent Kit for Multiplexed Samples). 300,000 fixed nuclei per mouse sample was used for the GEM-X Flex probe hybridization step. Probe hybridization was conducted using the GEM-X Flex Mouse Transcriptome Probe Kit, 16 Samples. After overnight probe hybridization 4 separate pools containing 4 samples each were generated for a total of 16 samples. Each pool was run across 1 GEM (Gel Bead in Emulsion) reaction for a total of 4 GEM reactions. After GEM generation and barcoding, GEMs were recovered and the resulting material underwent Pre-Amplification and SPRI bead cleanup. For each pool of 4 samples, we generated 1 GEM-X Flex Gene Expression library for a total of 4 libraries. During the final library construction PCR step each library was uniquely indexed using the 10x Genomics Dual Index Plate TS Set A. After the final library construction PCR and SPRI bead size selection, the final GEM-X Flex Gene Expression libraries underwent BioAnalyzer QC (Agilent High Sensitivity DNA Kit). All libraries contain the standard Illumina paired-end constructs that start and end with P5 and P7 sequences. Read 1 encodes the 16 bp GEM barcode and the 12 bp UMI. Read 2 encodes the ligated probe insert, a constant sequence, and the 8 bp Probe Barcode sequence that identifies the mouse probe set used in the hybridization step.