RNA extraction was performed using the RNeasy Plus Mini Kit (Qiagen #74134). Total RNA was subjected to alkylation by iodoacetamide (10mM, Sigma #I1149) for 15 min. 500ng alkylated RNA was used as input for generating 3'-end mRNA sequencing libraries using a commercially available kit (QuantSeq 3_mRNA-Seq Library Prep Kit FWD for Illumina, Lexogen #015.24). We then performed a Nextera XT library prep according to Illumina protocol 15031942 Rev. E. We quantified the libraries by qubit DNA HS and performed profile analysis by TapeStation 4200. We performed single end 75nt sequencings on NextSeq 500.
GEO Accession
GSM4456425