GSM4309361: b2_Mix3_5_540; Homo sapiens; OTHER
not extracted, tempoSeq lysis buffer used to lyse cells, which were then frozen and 2ul used for TempO-Seq library building [Assay Principle]  The TempO-Seq assay utilizes a detector oligo (DO) pool specific to the RNA targets to be monitored. BioSpyder offers fixed content and custom DO pools to measure focused sets of transcripts, up to whole transcriptomes. The assay can utilize purified RNA, cell lysates, tissue lysates, or FFPE tissue lysates (FFPE kit available separately). In addition, both positive and negative control samples can be included. Samples are annealed to a matched pair of detector oligos (DOs), with each pair targeting a single gene. The DO pool targets up to tens of thousands of RNA sequences, depending on the targeted gene content of the assay, but all share the same pair of primer binding sequences (indicated in Fig 1. by the red and yellow sections of each DO). The DOs hybridize to sequences of the targeted RNA molecule such that their respective 3' and 5' ends are adjacent. Each UDO has an additional target-specific sequence at their 3' end that hybridizes to the targeted RNA. After DO annealing, excess oligos and mis-hybridized or weakly hybridized DOs are removed by nuclease digestion, and the annealed oligos are ligated to form amplifiable templates. The ligated DOs are amplified with one pair of primers (making each PCR reaction single-plex regardless of the number of genes measured), with a different combination of sample index/tag sequences assigned to each sample. The PCR products are pooled together into a sequencing library, which is then purified and run on an Illumina NGS to count the number of ligated detector oligo sequences per sample (ThermoFisher-compatible kit under development). Because TempO-Seq includes an end-point PCR reaction, quantitation of the amplified products before library pooling is not needed. Very low concentration and negative control samples are expected to show low reads in the sequencing data. [Library construction protocol]  BioSyder -TempO-Seq protocol v2.1 sept 6,2017 Human S1500, index E,F,H,G were used for indexing