title: GSM8897533: GAT, HGC, TERM, Mouse 4; Mus musculus; ncRNA-Seq
library_construction_protocol: LAR mice were anesthetized with gas anesthesia and euthanized by means of cardiac puncture blood draw (exsanguination) and double thoracotomy. The liver was removed, weighed, dissected into individual lobes (1- left/right medial, 2- left lateral and 3- remaining lobes) and snap frozen on liquid nitrogen. All tissues were collected and frozen within 10-16 minutes of thoracotomy procedure. All groups were treated identically. Organ samples were maintained at -80C or lower temperatures throughout the storage, and during transportation and shipment. TERM mice were anesthetized with ketamine/xylazine/acepromazine at a dose of 120/15/3 mg/kg and euthanized by means of cardiac puncture blood draw (exsanguination) and double thoracotomy. The spleen was dissected fresh from each carcass and placed in a tube containing RNA Later. The carcass was then wrapped in foil and frozen in MELFI on board the ISS or in ground -80C freezers. Carcasses were maintained at -80C or lower temperatures throughout the storage on board the ISS, in ground freezers and during transportation and shipment. The mice were brought back to earth where dissection was held. It should be noted that carcasses frozen whole onboard the ISS were free-floating and thus in direct contact with air. This prevented them from snap freezing like samples collected on Earth which were placeddirectly in liquid nitrogen. Carcasses were thawed at room temperature one at a time (staggered)until the tissues were sufficiently pliable for dissection (approximately 60-90 minutes). Once thawed, multiple tissues were collected from each carcass within 30min of the first tissue collected. The liver was removed from each carcass, weighed, dissected into individual lobes (1-left/right medial, 2- left lateral and 3- remaining lobes) and snap frozen on liquid nitrogen.  The miRNeasy-96 protocol was followed for all samples with some modifications, as follows. Tissue Preparation: To avoid overloading the miRNeasy-96 columns, some frozen samples were trimmed to smaller sizes. All kidney, liver, and spleen samples were all trimmed to ~25mgs due to their high RNA content. Pancreas samples were trimmed to ~25mgs if they exceeded 45mgs. GAT, MAT, and SCAT samples >200mgs were trimmed to ~100mgs as samples of large size homogenize poorly in 96-well format, even though adipose tissues have relatively low RNA content. BAT and lung samples >100mgs were trimmed to ~50mgs, and thymi >50mgs were trimmed to ~25mgs. The diaphragm did not require trimming, and whole hemi-brains and whole hearts were homogenized by hand with the TissueRuptor in 1ml QIAzol per 50mgs tissue, since trimming samples in a reproducible way to capture the same organ subregion for each would be difficult. Bead homogenization: All samples, including those previously homogenized with the TissueRuptor, were randomized in 96-well format. Two 96-well plates were run simultaneously as follows. 96-well collection microtube plates were chilled on dry ice, and trimmed tissue samples were placed at the bottom of each well. One pre-chilled 5mm stainless steel bead was then placed on top of the tissue samples with bead dispenser. 700ul pre-chilled (4C) QIAzol was then added to each well, or 700ul QIAzol homogenate from the pre-homogenized heart and brain samples. Plates were immediately homogenized on the TissueLyser II for a total of four 5min runs at 25Hz, and plates were rotated and switched between the two arms of the TissueLyser II between each run. Plates were then left at room temperature for 5min before pelleting debris at 6000 x g for 1min at 4C. Phase Separation & RNA collection: Debris- and bead-free homogenate was then transferred to a new plate, to which 140ul chloroform was added to each well, and plates were shaken vigorously for 3min at room temperature before being spun at 6000 x g for 4min at 4C. 300ul aqueous phase was then transferred to a new S-Block, to which 525ul 100% EtOH was added and mixed by pipetting, followed by a room temperature spin at 6000 x g for 30s. The entire volume was then transferred to a miRNeasy-96 plate on top of an S-plate, sealed with AirPore tape sheet, and spun at 5600 x g for 4min at room temperature. Flow through was discarded, and columns washed with 800ul RWT and spun as above. This was repeated with 800ul RPE, and a final 800ul RPE wash was followed by a 10min spin. Finally, 50ul RNase-free water was added to each column and incubated at room temperature for 1min, then spun at 5600 x g for 4min at room temperature. The elution was repeated with a second volume of 50ul RNase-free water. RNA samples were stored at -80C. All samples were processed using the MGIEasy Small RNA Library Prep Kit with the high-throughput MGI SP-960 sample prep system according to the manufacturer's protocol to generate sequencing libraries.