GSM5850115: 381_36_mouse_37_postappl_day_14_NaCl; Mus musculus; miRNA-Seq
For total lung RNA preparation, the left lung was flash frozen in liquid nitrogen immediately after dissection. Frozen lungs were homogenized in 2 mL precooled RLT buffer (Qiagen, Hilden, Germany) + 1 % β-mercaptoethanol (Sigma-Aldrich) using the Peqlab Precellys 24 Dual Homogenizer and 7 mL-ceramic bead tubes (#91-PCS-CK28L, Peqlab, Erlangen). 150 µL homogenate were then mixed with 550 µL QIAzol Lysis Reagent (#79306, Qiagen). After addition of 140 µL chloroform (Sigma-Aldrich), the mixture was shaken vigorously for 15 sec and centrifuged for 5 min at 12,000 xg and 4 °C. 350 µL of the upper aqueous phase (containing RNA) were then further purified using the miRNeasy 96 Kit (#217061, Qiagen) according to the manufacturer's instructions. After purification, RNA concentration was determined using a Synergy HT multimode microplate reader and the Take3 module (BioTek Instruments, Winooski, VT, USA). RNA quality was assessed using the 2100 Bioanalyzer (Agilent Technologies).   cDNA libraries were prepared using the TruSeq Stranded Total RNA Sample Preparation Kit (Illumina, San Diego, CA, USA) and 200 ng of total RNA. Following purification and PCR enrichment of the cDNAs, the library was diluted to 2 nM and clustered on the flow cell at 9.6 pM, using the TruSeq SR Cluster Kit v3-cBot-HS (#GD-401-3001, Illumina) and the cBot instrument (Illumina). Sequencing of 52 bp single reads and seven bases index reads was performed on an Illumina HiSeq 2000 using the TruSeq SBS Kit v3-HS (#FC-401-3002, Illumina). Approximately 20 million reads were obtained per sample. For miRNA, the TruSeq Small RNA Library Preparation Kit was used (Illumina) to prepare the cDNA library: As a result of miRNA processing by Dicer, miRNAs contain a free 5'-phosphate and 3'-hydroxal group, which were used to ligate specific adapters prior to first and second strand cDNA synthesis. By PCR, the cDNAs were then amplified and indexed. Using magnetic Agencourt AMPure XP bead-purification (#A63881, Beckman Coulter), large DNAs were separated and small RNAs were enriched. Similar to mRNA, the samples were finally clustered at 9.6 pM and sequenced, while being spiked into mRNA sequencing samples. 