GSM5385772: 1657-A029924; Homo sapiens; miRNA-Seq
A total of 30uL of thawed plasma was co-incubated with 1:1 volumes of HTG Plasma Lysis buffer (pre-warmed to 50 degrees celsius) and 1/10th v/v proteinase K for three hours at 50 degrees celsius with shaking (200prm). The lysed sample was transferred to a standard 96-well micro-titer plate, referred to as the sample plate. Target capture was done by the HTG EdgeSeq chemistry. Briefly, the nuclease protection probes (NPPs) were added to the lysed samples in the sample plate in excess amount and hybridized to the target miRNA. Then S1 nuclease was added to digest non-hybridized mRNA and excess NPPs, thus producing a stoichiometric amount of target miRNA NPP duplexes. After the S1 digestion was completed, the processed sample was transferred to a new 96-well micro-titer plate with a v-bottom, referred to as the stop plate, and S1 digestion was terminated by the addition of termination solution followed by heat denaturation of S1 enzyme. Each processed sample from the stop plate was used as   template to set up PCR reactions with specially designed primers, referred to as tags. These tags share common sequences that are complementary to 5'-end and 3'-end “wing” sequences of the probes and common adaptors required for cluster generation on an Illumina sequencing platform. In addition, each tag contains a unique barcode that is used for sample identification and multiplexing. HTG has developed 12 forward and eight reverse primer tags; therefore, up to 96 samples can be analyzed simultaneously on an Illumina sequencing platform. Clean-up procedures were performed according using AMPure cleanup protocols for Illumina Sequencing Libraries. The library was quantified in accordance with the HTG EdgeSeq KAPA Library Quantification kit for Illumina Sequencing. All samples and controls were quantified in triplicate. No Template Control (NTC) reactions were made for each master mix used (using the same master mix as for samples) during the qPCR process. NTCs serve as a test for qPCR reagent contamination. Molecular-grade water is used in place of test sample in the NTC reactions using the same volume as the template.