GSM8676729: 4sU labeling, 1µM ETO, rep2; Homo sapiens; RNA-Seq
For single cell RNA sequencing, single cell suspension was prepared for 10X Genomics sample loading. For RNA-seq, RNA was extracted using RNeasy Mini Kit (Qiagen). For NOTE-seq, Libraries were constructed using Chromium Next GEM Single Cell 3ʹ Kit v3.1 user guide (Rev E) with serval additional steps detaied in the Method section. For regular 10xgenomics, libraries were constructed using  Chromium Next GEM Single Cell 3ʹ Kit v3.1 user guide (Rev E). For SLAM-seq, 2 µg RNA were combined with 10 mM iodoacetamide (Sigma-Aldrich, St. Louis, MO, USA), 50 mM sodium phosphate buffer (pH 8.0) and 50% DMSO, followed by 15 min of incubation at 50°C. The reaction was stopped by adding 20 mM DTT, followed by RNA purification using 1.8X RNA beads (Aline Bioscience) and RNA elution with 11 µl nuclease-free water. Then, 500 ng RNA was subject to library construction using Quant-seq 3′ mRNA-Seq Library Prep Kit (Lexogen)