GSM8147301: hepatocyte, ALAS12-KO, rep1 (miRNA-seq); Mus musculus; miRNA-Seq
Mice were deeply anesthetized with ketamine/xylazine (100/10 mg/kg, i.p.) and given a toe pinch test to ensure sufficient anesthetization. For whole liver experiments, mice were perfused with 1x phosphate-buffered saline (PBS) via the left cardiac ventricle, and liver tissues were harvested Hepatocyte isolations were performed using the two-step collagenase perfusion method (PMID: 6273298). In brief, a lateral incision was made through the integument and abdominal wall up to the ribcage. The diaphragm and pleural cavities were incised, a 25G i.v. catheter was inserted into the hepatic portal vein, and the inferior vena cava was cut to create an outlet in the circulatory system for exsanguination. Liver Perfusion medium (Gibco, 30 mL), heated to 37℃, was pumped through the catheter line using a Gilson Minipuls 2 peristaltic pump at a rate of 6.5 mL/min, followed by 30 mL of 37℃ Hepatocyte Wash medium containing Collagenase B (0.05% Collagenase B solution). Liver tissue was excised, transferred to a petri dish containing 5 mL of liver digestion buffer [0.05% Collagenase B solution diluted 10X in Hepatocyte Wash medium (Gibco)], and incubated for 10-13 min at 37℃. Digested liver tissue was filtered through a 70 µm cell strainer and then centrifuged at 50 x g for 5 min at 4℃ to isolate a hepatocyte-enriched pellet. The hepatocytes were washed three times using Hepatocyte Wash medium and centrifugation at 50 x g for 5 min at 4℃. One milligram of total RNA was used to generate small RNA libraries for each sample. After adding 1 μL of QIAseq TM miRNA Library QC Spike-Ins (QIAGEN) to the total RNA, a 3' linker was ligated to it in a reaction containing 100 mM 3' linker, T4 RNA Ligase 2, truncated KQ (NEB), 10% PEG8000, 1x RNA ligation buffer, and 20 U RNase OUT overnight at 17°C. The 48-57 nucleotide sized 3' linker-ligated small RNAs were purified using 12% Urea-PAGE gel with radio-labeled oligonucleotides serving as size-markers. Subsequently, the small RNA-linker hybrid was subjected to a 5' ligation reaction overnight at 17°C containing 100 mM 5' adaptor, 20% PEG8000, 1x RNA ligase buffer, 1 mM ATP, 20 U RNaseOUT, and 5 U T4 RNA ligase 1. The linker-ligated RNAs were then reverse transcribed, PCR amplified using 12 cycles of PCR with forward and Illumina index reverse primers, and purified by 8% non-denaturing acrylamide gel. Sequencing was performed with HiSeq 4000 in SE50 mode.