GSM4173655: Trachea rep3 [S13_Trachea]; Homo sapiens; RNA-Seq
To obtain total RNA, cultured fibroblasts were trypsinized and harvested at a density no less than 1 X 106. Cells were then neutralized with fresh media and centrifuged at 1000 rpm for 10 min to retrieve the cell pellet. Total RNA was purified from cell pellet samples using the RNeasy Mini Kit (Cat No. 74106) (QIAGEN) according to the manufacturer's protocol. RNase-free DNase (QIAGEN) was treated on the column for 15 min to remove the minimum genomic DNA contamination. Quantification of all total RNA samples, initially, were performed using a NanoDrop 1000 spectrophotometer (Thermo Scientific), and integrity of samples were confirmed with the following three criteria for inclusion: (1) a concentration > 40 ng/ml, (2) an A260/A280 rating of 1.8 and 2.1, and (3) an A260/A230 ratio > 1.8. Total RNA submitted to the University of Wisconsin-Madison Biotechnology Center was verified for purity and integrity via the NanoDrop One Spectrophotometer and Agilent 2100 BioAnalyzer, respectively. Samples that met the Illumina sample input guidelines were prepared according the TruSeq® Stranded mRNA Sample Preparation Guide (Rev. E) using the Illumina® TruSeq® Stranded mRNA Sample Preparation kits (Illumina Inc., San Diego, California, USA). For each library preparation, mRNA was purified from 1μg total RNA using poly-T oligo-attached magnetic beads. Subsequently, each poly-A enriched sample was fragmented using divalent cations under increased temperature. The fragmented RNA was synthesized into double-stranded cDNA using SuperScript II Reverse Transcriptase (Invitrogen, Carlsbad, California, USA) and random primers for first strand cDNA synthesis followed by second strand synthesis using DNA Polymerase I and RNAse H for removal of mRNA. Double-stranded cDNA was purified by paramagnetic beads (Agencourt AMPure XP beads, Beckman Coulter). The cDNA products were incubated with Klenow DNA Polymerase to add an 'A' base (Adenine) to the 3′ end of the blunt DNA fragments. DNA fragments were ligated to Illumina adapters, which have a single 'T' base (Thymine) overhang at their 3′ end. Adapter-ligated DNA products were purified by paramagnetic beads, successively amplified in a Linker Mediated PCR reaction (LM-PCR) for nine cycles using Phusion™ DNA Polymerase and Illumina's PE genomic DNA primer set and then purified by paramagnetic beads. Quantity and quality of completed libraries were evaluated using a Qubit® dsDNA HS Assay Kit (Invitrogen, Carlsbad, California, USA), and an Agilent HS DNA or DNA1000 chip (Agilent Technologies, Inc., Santa Clara, CA, USA), respectively. Libraries were standardized to 2 nM. Cluster generation was executed using standard Cluster Kits (v4) and the Illumina cBot. Single end, 100 bp sequencing was completed using standard SBS chemistry (v4) on an Illumina HiSeq2500 sequencer. Images were analyzed using the standard Illumina Pipeline, version 1.8.2.
GEO
GSM4173655
GEO Accession
GSM4173655