title: GSM1948546: K52o_Dox_2; Homo sapiens; RNA-Seq
library_construction_protocol: Total RNA was extracted using the RNeasy Mini Kit (Qiagen) including on-column DNase digestion. Poly-A RNA was isolated from total RNA using the NEBNext Poly(A)mRNA Magnetic Isolation Module (E7490). For 4-SU seq, nascent RNA transcripts were labelled for 10 min using 1 mM 4-thiouridine (4sU, Sigma T4509). After RNA isolation the 4SU-labelled RNAs were biotinylated and enriched using MyOne Streptavidin beads (Life Technologies). Eluted RNAs were subsequently used for library preparation using NEBNext RNA-Sequencing kits. Libraries for the RNA-seq samples were constructed using the NEBNext Library Prep Kit (6100) following the instruction manual. Briefly, poly-A RNA was fragmented to generate 200 nucleotides long fragments. First and second strand cDNA synthesis was performed and the resulting cDNA was end-repaired, ligated to NEBNext adaptors. The cDNA was size selected using agarose gels and amplified by PCR. For the resulting RNA-seq libraries, amplicon sizes and quantities were determined using the Biorad Experion system. Libraries were sequenced on an Illumina NextSeq 500 following the manufacture’s instructions.
attributes.tag: GEO Accession
attributes.value: GSM1948546