Comment[GEAAccession] E-GEAD-312 MAGE-TAB Version 1.1 Investigation Title RNA-seq analysis of fractionations in human dermal fibroblast (Set1-directional RNA-seq-Fractionation)) Experiment Description To elucidate functions of lncRNAs, we isolated fractionation samples in human dermal fibroblast cell lines. After the isolation, we obtained gene expression profiles with the directional RNA-seq method. Experimental Design cell component comparison design Experimental Factor Name fraction Experimental Factor Type fraction Person Last Name Kasukawa Bono Person First Name Takeya Hidemasa Person Affiliation RIKEN Person Roles submitter submitter Public Release Date 2019-07-22 Protocol Name P-GEAD-188 P-GEAD-189 P-GEAD-190 P-GEAD-191 P-GEAD-192 P-GEAD-193 Protocol Type sample collection protocol nucleic acid extraction protocol nucleic acid library construction protocol nucleic acid sequencing protocol normalization data transformation protocol high throughput sequence alignment protocol Protocol Description Trypsinized cells were washed and lysed using cold lysis buffer containing 0.15% Igepal CA-630, 10 mM Tris pH 7.5, 150 mM NaCl. The lysate was 24 centrifuged in a sucrose cushion, after which the supernatant was taken as the cytoplasmic fraction. The nuclear pellet was washed once in buffer containing 20mM HEPES pH 7.5, 50% glycerol, 75 mM NaCl, 1 mM DTT, 0.5 mM EDTA and suspended again in the same buffer. An equal volume of nuclear lysis buffer containing 20mM HEPES pH 7.5, 300mM NaCl, 1M Urea, 1% Igepal CA-630, 10mM MgCl2, 1mM DTT, 0.2mM EDTA was added and incubated on ice for 5 min. After centrifugation, the supernatant was considered as the nucleoplasmic fraction and the pellet as the chromatin fraction. The chromatin pellet was washed once in buffer containing 10 mM HEPES pH 7.5, 10 mM KCl, 10% glycerol, 340 mM sucrose, 4 mM MgCl2, 1 mM DTT and suspended in the same buffer. RNA from each fraction was isolated using Trizol LS (Invitrogen) according to manufacturer's instructions. The libraries are prepared in accordance with the manufacturer's protocol using Illumina TruSeq Stranded Total RNA Library Prep Kit. The first step involves the removal of ribosomal RNA (rRNA) from total RNA using biotinylated, target-specific oligos combined with Ribo-Zero rRNA removal beads. The Ribo-Zero Human/Mouse/Rat kit depletes samples of cytoplasmic rRNA. Following purification, the RNA is fragmented into small pieces using divalent cations under elevated temperature. The cleaved RNA fragments are copied into first strand cDNA using reverse transcriptase and random primers, followed by second strand cDNA synthesis using DNA Polymerase I and RNase H. These cDNA fragments then have the addition of a single 'A' base and subsequent ligation of the adapter. The products are purified and enriched with PCR to create the final cDNA library. Libraries were subjected to 50-base single-end sequencing using an Illumina HiSeq 2500 instrument. Expression tables are normalized in FPKM and TPM. Tags were mapped to human genome assembly hg38 using TopHat 2.0.12. SDRF File E-GEAD-312.sdrf.txt Comment[AEExperimentType] RNA-seq of non coding RNA Comment[BioProject] PRJDB7993 Comment[Last Update Date] 2019-08-14