GSM6126572: Tag2F10_Tag02; Mus musculus; RNA-Seq
18 days after LPC injection, mice were deeply anesthetized and perfused with cold HBSS between 9am-11am (to decrease circadian fluctuations). Each brain was removed and under a dissection microscope individually micro-dissected; gray matter was isolated from the frontal cortex and white matter was isolated form optic tract, medial lemniscus and corpus callosum (attached gray matter and choroid plexus were carefully removed). Single cells were isolated using GentleMacs with the Neural Tissue Dissociation Kit (Papain) (Miltenyi Biotec). We added actinomycin D to a final concentration of 45 mM into the dissociation solution and enzyme mix to prevent ex-vivo transcription. The dissociated cell suspension was passed through a 70 mm cell strainer before labeling. Subsequently, cells were blocked with mouse FcR-blocking reagent (CD16/CD32 Monoclonal Antibody) and then stained for with the antibodiy against CD11b (PE/Cy7,M1/70, eBioscience, Cat:48-0451-82,1:200) and afterwards washed with PBS (Sigma, D8537). Cells were then stained with DAPI (4',6-diamidino-2-phenylindole, 1:10000 dilution; Sigma)  and viable (DAPI-negative) single CD11b-positive cells were sorted by flow cytometry (SH800; Sony) into 96 well plates filled with 4 mL lysis buffer containing 0.05% Triton X-100 (Sigma) and, ERCC spike-in Mix (1:24000000 dilution), 2.5 mM oligo-dT, 2.5 mM dNTP and 2 U/mL of recombinant RNase inhibitor (Clontech). The 96-well plates containing the sorted single cells were thawed and incubated for 3 min at 72°C and thereafter immediately placed on ice. To perform reverse transcription (RT) we added into each well a master mix of 0.59 μL H2O, 0.5 μL SMARTScribe™ Reverse Transcriptase (Clontech), 2 μL 5x First Strand buffer, 0.25 μL Recombinant RNase Inhibitor (Clontech), 2 μL Betaine (5 M Sigma), 0.5 μL DTT (100 mM) 0.06 μL MgCl2 (1 M Sigma), 0.1 μL Templateswitching oligos (TSO) (100 μM AAGCAGTGGTATCAACGCAGAGTACrGrG+G). Next RT reactions were incubated at 42°C for 90 min followed by 70°C for 5 min and 10 cycles of 50°C 2 min, 42°C 2 min; ending with 70°C for 5 min for enzyme inactivation. Preamplification of cDNA was performed by adding 12.5 μL KAPA HiFi Hotstart 2x (KAPA Biosystems), 2.138 μL H2O, 0.25 μL ISPCR primers (10 μM, 5' AAGCAGTGGTATCAACGCAGAGT-3), 0.1125 μL Lambda Exonuclease under the following conditions: 37°C for 30 min, 95°C for 3 min, 23 cycles of (98°C for 20 sec, 67°C for 15 sec, 72°C for 4 min), and a final extension at 72°C for 5 min. Libraries were then cleaned using AMPure beads (Beckman-Coulter) cleanup at a 0.7:1 ratio of beads to PCR product. Libraries were assessed by Bio-analyzer (Agilent 2100), using the High Sensitivity DNA analysis kit, and quantified using Qubit's DNA HS assay kits and a Qubit 4.0 Fluorometer (Invitrogen, LifeTechnologies). Further selection of samples was performed via qPCR assay against ubiquitin transcripts Ubb77 (primer 1 5'-GGAGAGTCCATCGTGGTTATTT-3' primer 2 5'-ACCTCTAGGGTGATGGTCTT-3', probe 5'-/5Cy5/TGCAGATCTTCGTGAAGACCTGAC/3IAbRQSp/-3') measured on a LightCycler 480 Instrument II (Roche). Samples were normalized to 160 pg/μL. Sequencing libraries were constructed by using in-house produced Tn5 transposase. Libraries were barcoded, pooled and purified in 3 rounds of AMPure bead (Beckman-Coulter) cleanup at a 0.8:1 ratio of beads to library. Libraries were then sequenced with 100 bp paired-end sequencing on DNBSeq platform (BGI group).