title: GSM9491716: BMDM, 1st Phase, Healthy3; Mus musculus; RNA-Seq
library_construction_protocol: This in vitro model was adapted from Edgar et al.(Edgar et al. 2021) to mimic diabetic-trained and untrained BMDM states. Male C57BL/6J mice (8 weeks old) from the Laboratory Animal Center of National Bioindustry (Chongqing, China) were randomly allocated to control or diabetic groups. For diabetes induction, mice received low-dose streptozotocin (42-45 mg/kg/d; Solarbio, China) via intraperitoneal injection after a 12h fast, repeated daily for 5 days. One week post-final injection, mice with nonfasted blood glucose >16.9 mmol/L were deemed type 1 diabetic. After 6 weeks of hyperglycemia, bone marrow progenitors from both groups were isolated and cultured in 5 mmol/L glucose DMEM with recombinant murine M-CSF for 7 days (rest phase). Total RNA was extracted from tissues using TRlzol (Invitrogen, Carlsbad, California,USA) according to manual instruction. For cell samples, the TRlzol was directly added to the cell sample with the amount 1mL per 1x10^6 cells, followed by vortex mixing and standing for 5-minute. Themixture was centrifuged at 4°C at 12,000xg for 5 minutes, and the supernatant wastransferred to a new EP tube for extraction. 300ul of chloroform/isoamyl alcohol(24:1) was added into the transferred supernatant and the mixture was vigorouslyvortexed to ensure thorough mixing. Then the mixture was centrifuged at 4°C and12,000xg for 8 minutes. Take the clear upper aqueous layer and repeat the aboveprocess one time. After centrifugation, the final upper aqueous layer was carefully transferred to a new 1.5ml EP tube with a 2/3 supernatant volume of isopropanol(for low amount of tissues/ cell, 2ul 5mg/mL glycogen can be added forprecipitation), gently inverted to mix well, placed in a -20°C refrigerator for 2 hours.Next, the precipitation mixture was centrifuged with 17500xg at 4°C for 25 minutes.The supernatant was discarded, and the precipitation was washed with 0.9ml of 75%ethanol. The precipitation was suspended by inverting the tube up and downseveral times. Then the precipitation was collected by centrifuging at 17500g for 3minutes at 4°C and discarding the supernatant. Repeat the centrifugation to removesupernatant completely and the precipitation was dried in the biosafety cabinet for3-5 minutes. Finally, 20uL~200ul of DEPC-treated or RNase-free water was added todissolve the RNA. Subsequently, total RNA was qualified and quantified using Qseq-400(Bioptic, Taiwan, China). (1) Cell Lysis: Lyse the cells and keep the cells on the ice all the time. (2) DNA Transposition and Tagmentation: Add Tn5 transposase to the cell suspension after cell lysis and then purify it with Qiagen MinElute PCR Purification Kit. (3) DNA Fragments Enrichment: Determine PCR cycles based on the DNA concentration and then purify the products with Qiagen MinElute PCR Purification Kit. (4) Size Selecting: Select DNA fragments with agarose gel electrophoresi. Dissolve the products after gel extraction with elution buffer. (5) Library quality control; (6) Library circularization; (7) The library was amplified to make DNA nanoball (DNB); (8) Sequencing on DNBSEQ (DNBSEQ Technology) platform.