Illumina HiSeq 2500 paired end sequencing; Single-nucleus transcriptomics reveals functional compartmentalization in syncytial skeletal muscle cells
Single-nucleus transcriptomics reveals functional compartmentalization in syncytial skeletal muscle cells
For each sorting, we pooled two TA muscles from two mice. Isolated TA muscles were cut into small pieces, resuspended in 1ml hypotonic buffer (250mM sucrose, 10mM KCl, 5mM MgCl2, 10mM Tris-HCl pH 8.0, 25mM HEPES pH 8.0, 0.2mM PMSF and 0.1mM DTT supplemented with protease inhibitor tablet from Roche), and then transferred to 'Tissue homogenizing CKMix' tube (Bertin instruments) containing ceramic beads of mixed size. After incubating on ice for 15 minutes, samples were homogenized with Precellys 24 tissue homogenizer (Bertin instruments) for 20 seconds at 5,000 rpm. Homogenized samples were passed once through 70µm filter (Sysmex), twice through 20µm filter (Sysmex), and lastly though 5ml filter cap FACS tube (Corning 352235). DAPI (Sigma) was added to final concentration of 300nM to label DNA. GFP and DAPI double positive nuclei were sorted using ARIA Sorter III (BD).  For library generation, we used the Cel-Seq2 protocol as described by Hashimshony et al (4). 96-well plates were prepared using an automated pipetting system (Integra Viaflo). Each well contained 1.2µl of master mix (13.2µl 10% Triton X-100, 25mM dNTP 22µl, ERCC spike-in 5.5µl and ultrapure water up to 550µl total) and 25ng/µl barcode primers. Plates were stored in -80°C until use.  After sorting nuclei into the wells, plates were centrifuged at 4,000g for 1 minute. Plates were incubated on 65°C for 5 minutes and immediately cooled down on ice. First strand synthesis was performed by adding 0.4µl 5X first strand buffer, 0.2µl 0.1M DTT, 0.1µl RNase inhibitor (RNase OUT) and 0.1µl Superscript II per well (all ThermoFisher); 1 hour at 42°C and 15 minutes at 70°C. Second strand synthesis was then performed by adding 2µl of the RT product, 7µl ultrapure water, 2.31µl 2nd strand buffer, 0.23µl 10mM dNTP, 0.08µl  E.coli DNA ligase, 0.3µl E.coli DNA polymerase I and 0.08µl RNase H per well (all ThermoFisher); 2 hours at 16°C. All products from one well were pooled into one tube (~1110µl total). Pre-warmed AMPure XP (Beckman Coulter) was diluted 6X in bead buffer, and 1.322ml of diluted beads was added to the pooled sample. To facilitate clean-up, the samples were divided into four 1.5ml tubes, and incubated at RT for 15 minutes. Tubes were placed on magnetic stand for 5 minutes. Supernatants were discarded, and beads were washed twice with 700µl 80% Ethanol (30 seconds while the beads are attached to the magnet). Beads were air dried for 15 minutes, resuspended thoroughly in 8µl ultrapure water, incubated for 2 minutes on RT, and place on magnetic stand for 5 minutes. 6.4µl of supernatants were transferred to a new tube. The beads were resuspended once again in 6.4µl water, and 6.4µl supernatants were further transferred.   Next, in vitro transcription was performed by mixing 12.8µl cDNA, 3.2µl 10X T7 buffer, 3.2µl T7 polymerase and 3.2µl of each NTPs (MEGAscript T7 kit from ThermoFisher); 37°C for 13 hours. Transcription products were transferred to ice. To remove primers, 12µl EXO-SAP (ThermoFisher) was added for 15 minutes at 37°C. To fragment the RNA, 44μl aRNA and 5µl fragmentation buffer were added to the samples, and incubated for 1 minute at 94°C. Samples were immediately moved to ice and 5.6μl fragmentation stop buffer was added. 100μl pre-warmed RNAClean XP beads (Beckman Coulter) was added to the samples, and incubated on RT for 10 minutes. Samples were washed with 200μl 80% Ethanol three times using magnetic stand, air dried and resuspended in 7μl ultrapure water. After 2 minutes incubation, tubes were placed in magnetic stand for 5 minutes, and supernatants were transferred to new tubes. Until further use, samples were stored at -80°C. 1μl of RNA was analyzed on Bioanalyzer RNA pico chip for quality check. 5μg RNA diluted in 5μl ultrapure water was subjected to reverse transcription using 1μl random hexamer primer in 10.5μl total using Superscript II kit (ThermoFisher). Each RT products were PCR amplified using Phusion DNA polymerase (ThermoFisher) in 40μl total. In this reaction, 2μl RP1 and RP1X primers (uniquely indexed PCR primer) was used per reaction. Using AMPure 50μl XP beads and 200μl 80% Ethanol washing, PCR products were cleaned and dissolved into 25μl ultrapure water. Bead clean-up was repeated, but adding 25μl beads and eluting in 12μl water at the end. DNA concentration was measure with Qubit using high sensitivity reagent. 1μl sample was ran on Bioanalyzer using a high sensitivity DNA chip to measure size distribution, which we confirmed peaks around 400bp.