GSM8222203: Cup.rep1.Pos.Plate1.C11; Mus musculus; RNA-Seq
Microglia (myeloid cells) extraction was carried out on ice following a published protocol (Bennett et al. PNAS. 2016) through douncing, MACS myelin removal and FACS staining. Single live cells in the CD45lowCD11b+ (microglia) gate were sorted into 96-well plates for library preparation. Sorted microglia plates were prepared for scRNA-sequencing following the previously published Smart-seq2 protocol (Zhou et al 2019). Plates were thawed and primer annealing was performed at 72°C for 3 minutes.  Reverse transcription was performed by adding 6uL of reverse transcription mixture (95U SMARTScribe Reverse Transcriptase (100U/uL, Clontech 639538), 10U RNase inhibitor (40U/uL), 1XFirst-Strand buffer, 5mM DTT, 1M Betaine, 6mM MgCl2, 1uM TSO (Rnase free HPLC purified) to each well (PCR protocol - at 42°C for 90 min, followed by 70°C, 5 min). DNA preamplification was done by adding 15 uL of PCR amplification mix (1X KAPA HIFI Hotstart Master Mix (Kapa Biosciences KK2602), 0.1uM ISPCR Oligo (AAGCAGTGGTATCAACGCAGAGT), 0.56U Lambda Exonuclease (5U/uL, New England BioLabs M0262S) to each well (PCR protocol - (1) 37°C 30 min; (2)95°C 3 min; (3) 23 cycles of 98°C 20 s, 67°C 15 s, 72°C 4 min; (4) 72°C 5 min). cDNA was cleaned using PCRClean DX beads (0.7:1 ratio, Aline C-1003-50) and resuspended in 20uL EB buffer. Nextera XT DNA Sample Prep Kit (Illumina FC-131-1096) was used at 1/10 of recommendation volume, with the help of a Mosquito HTS robot for liquid transfer. Specifically, tagmentation was done in 1.6uL (1.2uL Tagment enzyme mix, 0.4uL cDNA) at 55°C, 10 min. To stop the reaction, neutralization buffer was added 0.4uL per well and incubated at room temperature for 5 min. Then 0.8uL Illumina 10-bp dual indexes (0.4uL each, 5uM) and 1.2uL PCR master mix were added to amplify whole transcriptomes using the following program: (1) 72°C 3 min; (2) 95°C 30 s; (3) 10 cycles of 95°C 10 s, 55°C 30 s, 72°C 1 min; (4) 72°C 5 min. Libraries from a single 384 plate were pooled together in an Eppendorf tube and purified twice with PCRClean DX beads. The quality and concentrations of the final mixed libraries were measured with Bioanalyzer and Qubit, respectively, before Illumina Nova sequencing. To maximize PCR amplification efficiency for cuprizone demyelination samples, biotinylated-oligo-dT30VN oligo and biotinylated-TSO oligo were used in place of Oligo-dT30VN and TSO respectively, as described in an enhanced Smart-seq2 protocol (Gardeux et al 2022).