title: GSM4452039: MC-RR at 1000 μg/L [ES150]; Homo sapiens; RNA-Seq
library_construction_protocol: Cells were thawed in Qiazol lysis buffer (Qiagen, Hilden, DE) further homogenized (Bullet Blender Storm 24 mixer mill, Next Advance, Averill Park, NY) using the manufacturer's recommended settings.  Approximately 250 µl of chloroform:isoamyl alcohol (24:1; Sigma-Aldrich Chemical Co., St. Louis, MO) was added to each tube, mixed by inversion, and vortexed and incubated at room temperature for 3 min and transferred to a 1.5 ml Heavy Phase Lock Gel microfuge tube (5Prime, Inc., Gaithersburg, MD), incubated on ice (10 min) and centrifuged at 14,200 g (5 min, room temperature (RT)). The supernatant was transferred to a microcentrifuge tube (Eppendorf North America, Hauppauge, NY) and mixed with an equal volume of 70% ethanol and put on a RNeasy® MinElute Clean Up kit 2.0 ml column (Qiagen) and processed according to the manufacturer's protocol with DNase treatment (RNase-free DNase kit, Qiagen) prior to final cleanup and elution. RNA was quantified and quality was confirmed using an Agilent RNA 6000 Nano kit with a 2100 Bioanalyzer (Agilent Technologies, Inc., Wilmington, DE). Typical RIN scores were above 9.  RNA eluates were stored at -80 °C Sequencing libraries were prepared using the TruSeq Stranded mRNA Library Prep for NeoPrep kit (Illumina), following the manufacturer's protocol. An input of 100 ng total RNA was used for each sample. Additional resuspension buffer was added to each library to obtain a final total volume of about 24 µL. The concentration of each library was determined using either the KAPA Library Quantification Kit for Illumina Platforms (Kapa Biosystems) or the Qubit dsDNA HS Assay kit (Thermo Fisher Scientific).  Libraries were normalized to 10 nM and combined into pools of 8-16 samples. Dilutions of libraries for quantitation and pooling was done using 10 mM Tris-HCl, pH 8.5.
attributes.tag: GEO Accession
attributes.value: GSM4452039