GSM2772328: S99 SSEA4pos; Homo sapiens; RNA-Seq
SSEA4+ and SSEA4- cells were sorted from normal or IPF lung fibroblast cultures (passages 4-8) using a BD FACS ARIA III cell sorter. RNA was then extracted using miRNeasy micro kit (qiagen) and a minimum of 700 ng of total RNA was utilized for sequencing analysis. A minimum of 700 ng of total RNA was used in “Dynabeads® mRNA DIRECT™ Micro Purification Kit” (Ambion).  The libraries were prepared using the Ion Total RNA-Seq Kit v2 (Life Technologies).  Samples were then loaded into an Ion Torrent for amplification onto Ion Sphere Particles using Ion PI™ Template OT2 200 Kit v3 (Life Technologies), sequencing chemistry, Ion PI™ Sequencing 200 Kit v3 (Life Technologies) and sequencing Chip, Ion PI™ Chip Kit v2 (Life Technologies).  Samples were sequenced to at least 10 million reads.  Raw reads in FASTQ format were aligned to the UCSC human reference genome (hg19) using TOPHAT.  Gene expression was calculated using a gene transfer file (GTF) from UCSC genes and normalized read quantification as FPKM (Fragments Per Kilobase of transcript per Million fragments mapped) calculated with Cufflinks.