title: GSM9302161: 4sU labeling, IAA, scRNA-seq,; Homo sapiens; RNA-Seq
library_construction_protocol: 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 MeOH-4sU-10X, one million eHAP1 cells were fixed in 80% cold methanol and incubated for 30 minutes at –20 °C. For chemical conversion, iodoacetamide (IAA) was added to the fixed cell suspension to a final concentration of 10 mM, followed by incubation for 16 hours at room temperature on a rotator in the dark. Cells were then pelleted by centrifugation at 1000 × g for 5 minutes and resuspended in quenching buffer (PBS, 0.1% BSA, 1 U/µl SUPERase-In, 100 mM DTT) for 5 minutes at room temperature. After a second centrifugation, cells were resuspended in PBS containing 0.1% BSA and 1 U/µl SUPERase-In, passed through a 20 µm strainer, counted, and immediately loaded onto the 10 x Chromium system using the Chromium Next GEM Single Cell 3ʹ Kit v3.1.