title: GSM5683837: cGH_Male_liver; Mus musculus; RNA-Seq
library_construction_protocol: The following buffers were prepared fresh daily in advance and kept on ice for up to 2 h: 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; 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. Due to the high cost of 10x Genomics library preparation, we prepared a single sequencing library for each of the 6 treatment groups studied, each of which was comprised of single nuclei extracted from four individual mouse livers (biological replicates) for each of the 6 groups: vehicle-treated male liver, 27-hr TCPOBOP-treated male liver, vehicle-treated female liver, 27-hr TCPOBOP-treated female liver, untreated male liver (marked Male-1 in the figures), and continuous GH (cGH)-infused male liver. Thus, each of our snRNA-seq samples represent single cell gene expression profiles from a population of four mice, rather than a single individual, minimizing the impact of individual variation on the single cell and differential expression transcriptomic profiles and datasets obtained. To minimize premature tissue thawing and RNA degradation, a 50-100 mg piece of each liver was cut and stored in an Eppendorf tube on dry ice until ready for processing. For this protocol, all pipette tips used were RNase and DNase free (Cat. #76322-132, 76322-134, 76322-150, 76322-154, VWR).    The frozen pre-cut pieces of liver (from all livers in the group) were 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. BSA Wash 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 into a 50 ml conical tube on ice. Three additional washes of BSA Wash Buffer (1 ml) was used to rinse the homogenizer and pestles on ice, passed through the same 40 µm cell strainer and then added to the homogenized sample in the conical tube. The homogenizer and pestles were then washed with MilliQ water on ice three times before processing 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 three 1.5 ml Eppendorf tubes, which were centrifuged at 500g in a swinging bucket centrifuge for 5 min at 4° C to pellet the lysed cells. The supernatant was discarded and the nuclei were reconstituted in 1 mL BSA Wash Buffer, pooling all three tubes back into a single sample. Half of the sample, 500 µL, was transferred to a new 1.5 mL Eppendorf tube, and diluted with 500 µL BSA Wash Buffer before pelleting at 500g 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 on ice (Cat # 43-50020-03, PluriSelect).  The strained sample was transferred to LoBind 1.5 mL Eppendorf tubes (Cat # 022431021, Eppendorf) on ice, quantified using a Cellometer (Nexcelom) and diluted to a final concentration of 1.2 x 106 nuclei/mL in BSA Wash Buffer. Samples were kept on ice from this point until they were used for library generation. Libraries were constructed by the Boston University Single Cell Sequencing Core facility following the manufacturer's instructions for 10x Genomics Single Cell 3' Sequencing (Version 3 chemistry). Single nucleus RNA-seq (snRNA-seq)