title: GSM6855767: Liver, low, day7, rep1; Homo sapiens; RNA-Seq
library_construction_protocol: Cell lysates were prepared on ice, using on-chip lysis using lysis buffer (QIAGEN Buffer RLT with 1% Betamercaptoethanol). Briefly, 175 µL of the lysis buffer was added to each of upper and lower channels and allowed to incubate on ice for 1 min. Lysates were then quickly removed from the two channels, combined in RNAse-free cryotubes, and frozen at -80 °C until further processing. RNA was extracted from these samples using the Rneasy® Mini Kit (QIAGEN; Frederick, MD), according to manufacturer's instructions with minor modifications. Briefly, centrifugation was carried out at 10,000 x g and spun for 30s. On-column extraction was performed by incubating 30 µL RNase-free water directly on the spin column membrane for 2 min, followed by elution at 10,000 x g for 1 min. The eluate was run through the column again using the above centrifugation parameters and then stored at -80 °C for later analysis. The extracted samples were then quantified on a Qubit™ 4 Fluorometer using the Qubit™ RNA High Sensitivity (HS) Assay Kit following the recommended protocol in the Qubit Assay Kit User Guide. Further quantification and qualification was performed on a 2100 Agilent Bioanalyzer according to the Agilent RNA 6000 Nano Kit Guide. All samples with a RNA Integrity Number (RIN) higher than 8 were used in further analysis. Illumina Stranded Total RNA Prep Ligation with Ribo-Zero Plus Kit was used for library generation. The protocol and corresponding calculations were carried out following the Illumina Stranded Total RNA Prep Ligation with Ribo-Zero Plus Reference Guide with minor modifications. Centrifugation steps were at 280 x g for 1 min as opposed to the recommended 10 s due to plate centrifuge limitations. Library samples were then quantified using the Qubit 1X dsDNA High-Sensitivity (HS) Assay Kit and measured on a Qubit™ 4 Fluorometer, using manufacturer's recommended parameters. To assess the quality and quantity during the preparation steps, library samples were measured on a 2100 Agilent Bioanalyzer according to the Agilent High Sensitivity DNA Kit Guide.