title: GSM2427911: 18_14d RNA-Seq; Homo sapiens; RNA-Seq
library_construction_protocol: RNA-Seq: For mRNA sequencing, 100 ng input total RNA per sample were converted into cDNA libraries following the Illumina TruSeq® stranded total RNA sample preparation protocol with Ribo Zero Gold (cat RS-122-2301). The total RNA, once depleted from cytoplasmic and mitochondrial ribosomal RNA, was fragmented and converted into double-stranded cDNA. Single-stranded adaptors that include unique barcode sequences for sample identification were attached via ligation. The resulting molecules were amplified via polymerase chain reaction (PCR). The fragment size distribution of each library was quality-controlled using the Agilent Tapestation® 2200, and libraries were quantified using the Kapa® library quantification kit (Kapa Biosystems®, cat KK4835). The 20 libraries were normalized to 2 nM and pooled in 4 sets as per the randomization plan for each flow cell lane (4 lanes containing each 6 libraries). The pooled libraries were spiked in with 10% PhiX library and were bound to the surface of flow cells at equimolar concentrations of 13 picoM. Each template molecule was clonally amplified up to 1000-fold using a Cbot2® system (Illumina). Sequencing by synthesis was performed on the Illumina HiSeq2500®, using a strategy of 2x50bp paired-end sequencing with a minimal total read depth of 30 millions reads paired-end per sample after subtracting off-target sequences (residual ribosomal RNA, phiX, homopolymer repeats). After cell lysis and total RNA extraction in phenol/chloroform, the aqueous phase was processeded using the RNeasy® Mini Kit (QIAGEN) with DNase digestion as per the manufacturer’s instructions
attributes.tag: GEO Accession
attributes.value: GSM2427911