GSM9396626: vASC_SFRP4ko+Capan-1_GFP_1; Homo sapiens; RNA-Seq
After 7-days growth media was aspirated and cells were washed with PBS. Trypsin (0.25%) was used to seperate cells into single cell suspension. Cells were then sorted based on RFP+ or GFP+ expression using FACS. RNA from cells was isolated using the RNeasy Plus Mini Kit (Qiagen, #74104). RNA samples were submitted to Cancer Genomics Center at The University of Texas Health Science Center at Houston (CPRIT RP240610). For DNA depletion, the RNA samples were treated with DNase using RNeasy Mini Kit (#74104, Qiagen, Germany) and RNase-free DNase Set (#79254, Qiagen, Germany). The treated RNA samples were eluted in 10ul nuclease-free water. Total RNA was quality-checked using Agilent RNA 6000 Pico kit (#5067-1513) by Agilent Bioanalyzer 2100 (Agilent Technologies, Santa Clara, USA). RNA with DV200 greater than 66% was used for library preparation. Libraries were prepared with SMARTer Stranded Total RNA-Seq Kit v2 (#634418, Takara Bio, Japan) and Unique Dual Index Kit (#634752, Takara Bio, Japan) following the manufacturer's instructions. The quality of the final libraries was examined using Agilent High Sensitive DNA Kit (#5067-4626) by Agilent Bioanalyzer 2100 (Agilent Technologies, Santa Clara, USA). The libraries were pooled evenly and went for the paired-end 75-cycle sequencing on an Illumina NextSeq 550 System (Illumina, Inc., USA) using High Output Kit v2.5 (#20024907, Illumina, Inc., USA).