title: GSM8690478: TT-seq_Ctrl1; Homo sapiens; OTHER
library_construction_protocol: For 4-thiouridine (4SU) labeling, cells were treated with 1 mM of 4SU for 15 minutes. Cells were harvested with 4 ml of TRizol reagent and total RNA was isolated by conventional phenol:chloroform extraction method. Isolated RNA (200 µg) was mixed with 2 µg of yeast spike-in RNA in 100 µL of RNase-free water. The mixture was fragmentized and biotinylated to isolate 4SU labeled RNA. Biotinylated RNA was denatured at 65°C for 10 minutes and rapidly cooled on ice for 5 minutes. Subsequently, 100 µL of streptavidin MicroBeads were added and incubated for 15 minutes with rotation. The beads-conjugated RNA was captured using uColumn in a magnetic field. Purified newly transcribed RNA was eluted by adding 200 µL of 100 mM DTT solution. To clean up and concentrate the eluates, the Monarch PCR & DNA cleanup kit (New England Biolabs, #T1030) was used following the manufacturer's instructions. A total of 200 ng of purified RNA was used for library construction with the KAPA RNA HyperPrep Kit with RiboErase (KAPA Biosystems, #KR1351) and the KAPA Unique Dual-indexed Adapter Kit (KAPA Biosystems, #KR1736), as per the manufacturer's instructions. Libraries were pooled and treated with Illumina free adapter blocking reagent (Illumina, #20024145).