GSM4878023: TCPOBOP_Male_1 [G175-M4]; Mus musculus; RNA-Seq
The following buffers were prepared fresh daily and kept on ice for up to 2 hours: Base Solution, 10 mM Tris-Cl, pH 7.4, 146 mM NaCl, 1 mM CaCl2 21 mM MgCl2; Lysis Buffer, Base Solution containing 0.1% Triton X-100 (Cat. #T8787, Sigma), with 80 U/mL Protector RNase Inhibitor (Cat # 3335402001, Roche) added just prior to use; ST Nuclei Wash Buffer (ST Buffer), Base Solution containing 0.01% BSA (Cat. #SRE0036, Sigma), with 80 U/mL Protector RNAse Inhibitor added just prior to use; BSA Wash Buffer, 1X PBS containing 2% BSA and 0.02% Tween-20, with 80 U/mL Protector RNase Inhibitor added just prior to use. To minimize premature tissue thawing and RNA degradation, a ~250 mg piece of each liver was placed on dry ice and then cut into 2-3 smaller pieces, and stored in an Eppendorf tube on dry ice until ready for further processing. For this protocol, all pipette tips used were RNase-free and DNase-free (Cat. #76322 series, VWR). Each pre-cut liver sample was transferred to a 3 mL glass-on-glass dounce homogenizer with 1 mL of Lysis Buffer, on ice. Keeping the homogenizer on ice, the sample was dounced for 10 strokes with pestle A (loose fit) followed by ~10 strokes with pestle B (tight fit) until fully homogenized. Homogenization was performed in under one minute, while avoiding foaming and splattering of the sample. ST Buffer (1 ml) was then added, pipetted up and down a few times to mix, and the sample was then passed through a 40 µm cell strainer (Cat. # 10199-655, VWR) into a 50 mL conical tube on ice. A second 1 ml of ST Buffer was used to rinse the homogenizer and pestles, passed through the same 40 µm cell strainer and then combined with the homogenized sample. The homogenizer and pestles were then washed with Milli-Q water three times before being used to process the next sample. Homogenized samples were kept on ice until all samples were ready to proceed to the next stage. Each homogenized sample was divided into two 1.5 ml Eppendorf tubes, which were centrifuged at 500g in a swinging bucket centrifuge (Dynac Centrifuge, Clay Adams) for 5 min at 4° C to pellet the lysed cells. To avoid damage to the nuclei, a swinging bucket rotor was used to minimize the shear forces generated using a fixed-angle rotor. The supernatant was removed from the pelleted nuclei and the pelleted nuclei were gently resuspended in 1 ml BSA Wash Buffer while pooling the two tubes back into one sample, and then centrifuged at 500g in a swinging bucket rotor for 5 min at 4° C. The supernatant was discarded and the pellet was resuspended in 1 mL BSA Wash Buffer then passed through a 20 µm cell strainer (Cat # 43-50020-03, PluriSelect).  333 µl of strained sample was transferred to a LoBind 1.5 mL Eppendorf tube (Cat # 022431021, Eppendorf) on ice and 667 µl of Trizol LS reagent was immediately added to that tube, which was vortexed for a few seconds then stored at -20° C. Frozen samples were thawed on ice, and vortexed for 10 s to fully resuspend each sample. Chloroform (isoamyl alcohol free, 0.2 ml) was then added, followed by vortexing vigorously for 15 s. The sample was allowed to sit for 2-3 min at room temperature and then spun at 12,000 x g for 15 min at 4° C. The clear upper, aqueous phase was carefully transferred to a new centrifuge tube, being careful not to disturb the genomic DNA-containing interface. Isopropanol (0.5 ml) was added to each sample followed by vortexing for 10 s. Glycogen (Cat. #AM9510, ThermoFisher) was then added to each sample (1 µg per 20 µl reaction). Samples were vortexed and then incubated for 10 min at room temperature. Samples were centrifuged at 12,000 x g for 20 min at 4° C, and the supernatant was discarded. The RNA pellet was washed with 1 mL of 75% ethanol by vortexing, and followed by centrifugation at 7,500 x g for 5 min at 4° C. The ethanol wash was removed and sample left to air dry for 5-10 min. Final RNA pellets were resuspended in 20 µl Milli-Q water, quantified on a Qubit instrument using Qubit RNA HS Assay (Cat. #Q32852, Invitrogen) and stored at -20° C. Illumina sequencing libraries were prepared by poly(A) selection using the NEBNext Poly(A) mRNA Magnetic Isolation Module (Cat #E7490L) using 0.5 µg of input RNA, followed by cDNA synthesis and library preparation using the NEBNext Ultra Directional RNA Sequencing for Illumina kit (Cat #E7420L).