GSM8013357: Placental_Arteries_1, snRNAseq; Homo sapiens; RNA-Seq
Single-nuclei RNA sequencing (snRNA-seq) was performed on the three matched tissue types from four healthy pregnant women. 1-5 mg samples were processed using the 10X Genomics Chromium Nuclei isolation Kit (PN-1000494, 10X Genomics, UK). Tissue chunks were cut to 1 mm cubes on dry ice. Samples were transferred to dissociation tubes in 200ul lysis buffer and homogenized using the pestles with 30 strokes. Further 300ul lysis buffer was added and samples homogenized with 20 further strokes. Samples were incubated at 4oC for 15 minutes. Nuclei were isolated and purified following the manufacturer's instructions with minimal alterations. To avoid loss of nuclei, centrifugation times were extended to 15 minutes and only one wash was performed in resuspension buffer. For the final step, pellets were resuspended in approximately 50ul resuspension buffer.For nuclei counts, 10ul isolated nuclei were mixed with 10ul VitaStain AOPI Staining Solution (CS2-0106-5ml, Nexcelom) and counted using a Countess™ II FL automated cell counter (AMQAF1000, ThermoFisher Scientific, UK). For each samples, two full fields of view were imaged using brightfield, GFP and RFP wavelengths. Isolated nuclei were manually counted using Fiji Cell counter application (Schindelin et al 2012). The freshly prepared nuclei were further processed to sequencing libraries using 10X Genomics Chromium Next GEM Single Cell 3′ Kit v3.1 (PN-1000268, 10X Genomics, UK) following the manufacturer's protocol (CG000315 revC). Libraries were analysed using TapeStation HSD5000 (Agilent Technologies). sn-RNA sequencing