GSM6647601: P3_sample1, replicate 1, snRNAseq; Homo sapiens; RNA-Seq
frozen human ACP tissues were thawed on ice, minced into 1-mm3 pieces and homogenized in 1 ml cold Nuclei EZ Lysis Buffer (NEB) using a douncer (stroking ~10-20 times). Then, the homogenate was filtered through a 40-μm cell strainer and incubated in 2 ml of NEB for 5 minutes at 4 °C. Samples were spun for 5 minutes at 250-300 x g at 4 °C, and the pellet was resuspended in Nuclei Wash and Resuspension Buffer supplemented with DAPI. Nuclei suspensions were loaded into a MoFlo Astrios EQ Cell Sorter and collected in 1.5-ml tubes. Visium spatial gene expression slides and reagent kits for formalin-fixed paraffin embedded (FFPE) tissue were used according to the manufacturer's instructions (10x Genomics). Visium slides include 4 capture areas (6.5 x 6.5 mm), each with ~5,000 unique gene expression spots that are 55-m in diameter (with a 100 m center to center distance between spots). FFPE tissue blocks were prepared according to the manufacturer's instructions (tissue preparation guide CG000408). H&E images were prepared according to the protocol (Deparaffinization, H&E Staining, Imaging and Decrosslinking, CG000409). A spatial gene expression assay was performed according to protocol CG00407. SnRNA-seq libraries were acquired using the Chromium Single Cell V3.0 reagent kit following the manufacturer's protocol (10X Genomics). Each nuclear suspension containing 235, 616, 1002, 1110 and 1305 nuclei/μl was loaded into an independent lane. Libraries were sequenced on a NovaSeq6000 platform (Illumina) according to the manufacturer's instructions to generate 150-bp paired-end reads. Libraries were prepared with TruSeq Illumina libraries and sequenced on an Illumina NovaSeq 6000 system at a minimum sequencing depth of 25,000 read pairs per tissue-covered spot on the capture area by IntegraGen (Evry). Sequencing was performed with the recommended protocol (read 1: 28 cycles; i7 index read: 10 cycles; i5 index read: 10 cycles; and read 2S: 50 cycles). snRNA-seq