GSM7436973: HEK293T, pcDNA_ISG20_M1, rep1; Homo sapiens; RNA-Seq
Cells were lysed directly in the 10cm plate using 1mL of Trizol to extract total RNA. Total RNA was then depleted from ribosomal RNA using antisense DNA oligonucleotides and RNaseH treatment. Resulting rRNA-depleted RNAs were used as input for library preparation. Libraries were prepared following the protocol described in: DOI: 10.1093/nar/gku1235 Ligation of a 3' preadenylated adaptor(5' AppAGATCGGAAGAGCACACGTCTGAACTCCAddC 3'), followed by Reverse-transcription using a custom oligonucleotide (3' TTCTCGTGTGCAGACTTGAGGTCAGTG-SP18-TAGATGTGAGAAAGGGATGTGCTGCGAGAAGGCTAGA-NNNNNNNNATCACGGp–5'), circularisation of the obtained ssDNA and PCR amplification using a multiplexing forward PCR primer (5'-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT-3') and a multiplexing reverse primer containing an illumina index (5'- CAAGCAGAAGACGGCATACGAGATNNNNNNGTGACTGGAGTTCAGACGTGTGCTCTTCCG-3').