GSM7146879: lumbarSpine, gp120, scRNAseq; Mus musculus; RNA-Seq
We collected three lumbar spinal sections from each group and pooled them together for nuclei dissociation. To isolate nuclei for snRNA-seq, we used the detergent-mechanical cell lysis method. In brief, the lumbar spinal sections were placed in a pre-chilled Dounce homogenizer, and 500 μl of pre-chilled detergent lysis buffer was added. The detergent lysis buffer was prepared by adding 3µl of 20% Triton-X to 600 µl of low sucrose buffer (sucrose: 0.32 M; HEPES (pH = 8.0):10 mM; CaCl2: 5 mM; MgAc: 3 mM; EDTA: 0.1 mM; DTT: 1 mM). We homogenized the tissue first by 5 strokes with pestle A ('loose' pestle), then 5-10 strokes with pestle B ('tight' pestle) to prepare crude nuclei. We did not lift pestles out of the lysis buffer between strokes to minimize bubble creation during homogenization. We added 1 ml of low sucrose buffer to the Dounce homogenizer containing the crude nuclei and mixed gently by pipetting 2–3 times. We passed the crude nuclei preparation through a 40 µm strainer (Fisher scientific, cat. no. 08-771-1) into a pre-chilled 50 ml conical tube containing 1 ml of low sucrose buffer. Then, an additional 1 ml of low sucrose buffer was passed through the strainer, which brought the volume of the nucleus preparation to 3.5 ml (3 ml of low sucrose buffer and 500 μl of lysis buffer). We centrifuged the samples for 10 min (3,200g; 4 °C), and resuspended the pellets using 3 ml of low sucrose buffer by gently swirling to wash the pellet off the wall. The suspension was put on ice for 2 min and then transferred to an Oak Ridge tube. We homogenized the suspended nuclei for 30 s on ice using a homogenizer (IKA, Ultra Turrax T8; at setting 1). We carefully added 12.5 ml of density sucrose buffer (sucrose: 1 M; HEPES (pH = 8.0): 10 mM; MgAc: 3 mM; DTT: 1 mM) underneath the low sucrose buffer homogenate using a serological pipette to prevent creating any bubbles that could disrupt the density layers. We then centrifuged the Oak Ridge tube at 3,200 x g for 20 min at 4 °C. Once the centrifugation was complete, we immediately decanted the supernatant in a flicking motion. The nuclei remaining on the wall were resuspended with 500 µl of resuspension solution (BSA: 0.4 mg/ml; RNAse Inhibitor: 0.2 U/μl). The sample was then passed through a 40 μm Flowmi pipette tip filter (Sigma-Aldrich, cat no: H13680-004) Library was performed according to the manufacter's instructions (Chromium protocol CG000185 Rev). Briefly, nuclei were loaded onto the Chromium controller for library preparation and sequencing. GEMs were captured and underwent reverse transcription by incubating at in a thermal cycler. Next, GEMs were broken, and the sample cDNA was recovered and cleaned according to the instructions. Then 10 µl of purified cDNA sample was enzymatically fragmented and size-selected using SPRIselect magnetic beads. Illumina sequencing adapters were then ligated to the size-selected fragments and cleaned up using SPRIselect magnetic beads. Sample indices were selected and amplified, followed  by a double-sided size selection using SPRIselect magnetic bead