GSM8243015: 3D Kidney, Array-seq; Mus musculus; OTHER
10 µm fresh-frozen, OCT-embedded tissue sections on Array-seq slides were thawed at 37°C for 1 minute and fixed for 30-45 minutes at -20°C in 100% methanol. Following fixation, slides were dried for 1 minute at room temperature, and incubated with 2 U/µL Ribolock in 5X SSC for 5 minutes at room temperature. The SSC buffer was removed from the slide surface, sections were stained for 3 minutes at room temperature with hematoxylin, washed in nuclease-free water, and stained for 1 minute at room temperature with eosin diluted 1:10 in 0.45 M Tris-Acetic acid buffer, pH = 6.0. The slide was washed in nuclease-free water and kept at room temperature until dry and imaged at 20X magnification using an Olympus VS2000 slide scanner. Tissue sections were then permeabilized with 0.1% pepsin in 0.1 M HCl at 37°C for 15-20 minutes. After permeabilization, sections were washed once with 0.1X saline-sodium citrate (SSC) buffer. Tissue sections then underwent reverse transcription, tissue removal, exonuclease I treatment. cDNAs were eluted from the array by incubating for 10 minutes at room temperature in 0.1 M KOH and neutralized by adding 0.2X volume of 1M Tris-HCl. cDNAs were purified using a DNA Clean & Concentrator-5 kit and amplified using single-primer PCR using the KAPA HiFi HotStart ReadyMix kit. Amplified cDNAs were cleaned up using magnetic SPRISelect beads. 100 ng of amplified cDNA was tagmented using Tagment DNA TDE1 Enzyme and buffer for 5 min at 55°C and amplified by PCR using the Illumina Read1 primer and Illumina Read2-i7 reverse primers. Libraries were cleaned up using SPRISelect beads followed by gel purification using 2% E-Gel EX Agarose Gels and MinElute columns and quantified with the Qubit dsDNA High Sensitivity Assay Kit. Resulting libraries were sequenced on Illumina sequencing platforms.