GSM7656566: MEF_NT_rep2; Mus musculus; RNA-Seq
Total RNA was extracted using TRIzol reagent from cell lysates. Messanger RNA (mRNA) was isolated by oligo (dT) magnetic beads (NEB) and separated on a 15% polyacrylamide TBE-urea gel (Invitrogen). The mRNA at 50-100 nt was excised. The RNA was dephosphorylated by T4 PNK (NEB) followed by poly(A) tailing by PAP1 (NEB). After adapter ligation by T4 RNA ligase 2 truncated KQ (NEB), cDNA synthesis was performed by m-MLV-mut5. The synthesized cDNA was amplified by PCR using barcoded sequencing primers. The PCR products were separated on a 8% polyacrylamide TBE gel (Invitrogen). Expected DNA was excised and recovered from DNA gel elution.