title: GSM3731267: MCF7_3324_rep2_RNA; Homo sapiens; RNA-Seq
library_construction_protocol: The total RNA of cells was extracted using Trizol (Life Technologies) according to manufacturer's instructions. After DNase treatment, 5 μg of extracted RNA was depleted from ribosomal RNA using Ribo-Zero Gold Kit (Epicenter Madison, Winsconsin, USA). After fragmentation of the rRNA-depleted RNA, 500ng was reverse-transcribed using Super Script III Reverse Transcriptase (Invitrogen) and random primers (Invitrogen) following the manufacturer's instructions. Total RNA was extracted from cells using the QIAGEN RNeasy RNA extraction kit (QIAGEN, Netherlands), using on-column DNaseI treatment. Ribosomal RNA was removed using the riboZero rRNA removal kit (Illumina). RNA was then fragmented into 200bp fragments by incubation for 7.5 min at 95°C in fragmentation buffer (200 mM Tris-acetate, 500 mM Potassium Acetate, 150 mM Magnesium Acetate [pH 8.2]). First strand cDNA synthesis was performed using SuperScript III (Life Technologies), followed by synthesis of the second cDNA strand. Library preparation was performed using the KAPA hyperprep kit (KAPA Biosystems). Quality of cDNA and the efficiency of ribosomal RNA removal was confirmed using quantitative RT-PCR using the IQ Sybr Supermix, with primers for GAPDH, 18S and 28S rRNA.   Illumina library preparation was done using the Kapa Hyper Prep Kit. For end repair and A-tailing double stranded DNA was incubated with end repair and A-tailing buffer and enzyme and incubated first for 30 min at 20°C and then for 30 min at 65°C.Subsequently adapters were ligated by adding 30μl ligation buffer, 10 Kapa l DNA ligase, 5 μl diluted adaptor in a total volume of 110μl and incubated for 15 min at 15°C. Post-ligation cleanup was performed using Agencourt AMPure XP reagent and products were eluted in 20 μl elution buffer. After post-ligation cleanup, samples were incubated with 3 uL USER enzyme for 15 min at 37°C to digest the 2nd cDNA strand. Libraries were amplified by adding 25 μl 2x KAPA HiFi Hotstart ReadyMix and 5μl 10x Library Amplification Primer Mix and PCR, 10 cycles. Samples were purified using the QIAquick MinElute PCR purification kit and 300bp fragments selected using E-gel. Correct size selection was confirmed by BioAnalyzer analysis. Sequencing was performed using Illumina NextSeq 500 machines and generated 43bp single end reads.
attributes.tag: GEO Accession
attributes.value: GSM3731267