GSM9694774: Granuloma at 90 days_1; Mus musculus; RNA-Seq
Lung granulomas and/or lung tissue were rapidly dissected and immediately homogenized. Large granulomas were cut into smaller pieces on ice in the buffer using spring scissors before dounce homogenization. The homogenates were filtered (40 µm Flowmi filters, Bel-Art) and pelleted by centrifugation at 500 g for 5 minutes at 4C. The nuclei pellets were resuspended in homogenization buffer with 25% iodixanol (OptiPrep™ Density Gradient Medium, Millipore Sigma), and single nuclei were purified on an iodixanol gradient (25%-30%-40%) by centrifugation at 2,200 g for 20 minutes at 4C without brake. A 120µl aliquot containing nuclei was taken at the interface between the 30% and the 40% iodixanol layers. Nuclei concentrations were determined using a Countess 2FL and Trypan Blue staining. The nuclei suspensions were used directly for single-nucleus capture in the 10x Genomics Chromium iX. Single nuclei RNA-sequencing was performed using a Chromium Next GEM Single Cell 5' Reagent Kit, v2 on a Chromium iX, according to the manufacturer's instructions. Briefly, single nuclei suspensions, gel beads, and partitioning oil were loaded onto a Chromium NextGEM K Chip, for a target recovery of up to 10,000 nuclei per sample. The chip was loaded into a Chromium iX (10X Genomics) and single nuclei were captured in Gel Bead-In-Emulsions (GEMs). Reverse transcription was performed in a PCR cycler, and the cDNA was collected using Recovery Agent (10X Genomics), purified with DynaBeads MyOne Silane beads (10X Genomics), amplified with PCR for 13 cycles, and further purified with SPRISelect reagent (Beckman Coulter). Quality control and quantification of the amplified cDNA was performed on a Tapestation 4200 (Agilent) using D5000 High Sensitivity ScreenTapes (Agilent, 5067-5592). 5' single nuclei RNA-seq libraries were generated using 50ng of the amplified cDNA. After fragmentation, end-repair, A-tailing and size selection purification, the cDNA was ligated with Illumina adaptors and subsequently purified with SPRISelect reagent. Libraries were amplified and barcoded by PCR with dual index oligonucleotides from the Chromium Dual Index TT Set A kit (10X Genomics), for a total of 15 PCR cycles. The final PCR products were subjected to double-sided size selection with SPRISelect beads (0.6x and 0.8x). The library fragment size distribution was analyzed on the Agilent 4200 Tapestation, and library yields and concentrations were determined by qPCR using the KAPA Illumina Library Quantification PCR kit (Roche Diagnostics). Sequencing of the libraries (min. depth: 20,000 paired reads/nucleus) was performed on an Illumina NovaSeq instrument operated by a commercial provider, with the following paired-end sequencing settings: Read1, 151 cycles; i7 index, 10 cycles; i5 index, 10 cycles; Read2, 151 cycles.
GEO
GSM9694774_r1