GSM9471521: Mite, untreated, rep2,snRNA; Dermatophagoides farinae; RNA-Seq
Whole mites were cut into small fragments (∼0.1 cm) and suspended in a mixture of lysis buffer and PBS. After two washes with PBS, ice cold lysis buffer was added for 2 min on ice. Tissue was homogenized using a Dounce homogenizer, gently pipetted, and incubated on ice for 6 min. Lysis was stopped with chilled 4% BSA, followed by gentle trituration. Following centrifugation (300 g, 5 min, 4 °C), the pellet was resuspended in lysis buffer with 4% BSA, incubated on ice, and cleared of debris using a Debris Removal Solution. After another centrifugation, nuclei were resuspended in nuclear resuspension buffer (PBS with 2% BSA and 0.1% RNase inhibitor), pooled from three replicates, filtered through a 20 μm strainer, and counted. Single nucleus RNA seq libraries were prepared using the 10x Genomics Chromium Single Cell 3′ Kit (v3) and sequenced on an Illumina NovaSeq 6000 platform, yielding >20,000 paired end reads per nucleus.