title: GSM2257130: S63GT; Homo sapiens; miRNA-Seq
library_construction_protocol: RNA was collected using TRIzol® according to standard protocol: Initial RNA concentration and integrity was assessed using the Qubit 2.0 Fluorometer (Life Technologies Corp., Carlsbad, CA) and Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA). The sequencing library was constructed using the NEBNext Small RNA Library Prep Set and protocol companion (New England Biolabs, Ipswich, MA). Briefly, starting with 1 µg of total RNA, a 3’-tagging sequence (the 3’ Adaptor Oligo) is ligated to the 3’ end of the RNA. Small RNA with 3’ ends containing either a 2’,3’-OH or 2’-O-Me,3’-OH are efficiently tagged in the reaction. Excess 3’ Adaptor is removed using a reverse transcription primer. A 5’-tagging sequence (the 5’ Adaptor Oligo) is ligated to the 5’ end of 5’-monophosphorylated RNA. cDNA was synthesized using reverse transcriptase. The cDNA is then amplified by PCR using a standard forward primer and an indexing reverse primer. The library was then cleaned up using a Zymo DNA Clean & Concentrator-5 (Zymo Research, Irvine, CA). Size selection is performed on the library using the BluePippin (Sage Science, Inc., Beverly, MA) and the 3% agarose gel cassette (Sage Science, Inc.). To calculate final library concentration the quantity and average fragment length for each library was measured using the Agilent DNA 7500 Assay (Agilent Technologies, Santa Clara, CA), and Qubit High Sensitivity DNA Assay (Life Technologies Corp.) Libraries were normalized to 2nM in buffer EB (Qiagen, Santa Clarita, CA) and combined in equal molar amounts. Single read sequencing of 51bp was performed using the Illumina MiSeq platform and MiSeq Reagent Kit v2 (50 cycles) (Illumina, Inc.).  A total of 11.7 million single reads passing filter were attained.