title: GSM4712926: HCD34C-GFP; Homo sapiens; RNA-Seq
library_construction_protocol: Total RNA was isolated with E.Z.N.A.® Total RNA Kit (Omega Bio-tek, Norcross, GA) according to the manufacturer`s protocol. The RNA concentration was measured with Nanodrop 2000c spectrophotometer (Thermo Scientific Inc., Waltham, MA, USA) and integrity was assessed using Agilent 2200 Tapestation instrument (Agilent Technologies, Santa Clara, CA, USA). Libraries were prepared according to Illumina's instructions. One microgram of total RNA was used to prepare Ribo-Zero RNA-Sequencing (RNA-Seq) libraries.   Briefly, Ribosomal RNA (rRNA) is removed using biotinylated, target-specific oligos combined with Ribo-Zero rRNA removal beads following Illumina Reference Guide (Illumina, San Diego, CA). After purification, the RNA is fragmented into small pieces using divalent cations under elevated temperature. First strand cDNA syntheses were performed at 25°C for 10 minutes, 42°C for 15 minutes and 70°C for 15 minutes, using random hexamers and ProtoScript II Reverse Transcriptase (NEW ENGLAND BioLabs Inc.). In a second strand cDNA synthesis the RNA templates were removed, and a second replacement strand was generated by incorporation dUTP (in place of dTTP, to keep strand information) to generate ds cDNA. The blunt-ended cDNA was cleaned up from the second strand reaction mix with beads. The 3`ends of the cDNA were then adenylated and followed by the ligation of indexing adaptors. PCR (15 cycles of 98°C for 10 seconds, 60°C for 30 seconds and 72°C for 30 seconds) were used to selectively enrich those DNA fragments that have adapter molecules on both ends and to amplify the amount of DNA in the library.   The libraries were quantified and qualified using the High Sensitivity D1000 Screen Tape on an Agilent 2200 TapeStation instrument. The libraries were normalized, pooled and subjected to cluster and pair read sequencing was performed for 150 cycles on a HiSeq instrument (Illumina, Inc. San Diego, CA, USA), according to the manufacturer's instructions.
attributes.tag: GEO Accession
attributes.value: GSM4712926