GSM8220101: EAE,rep1,Neg,Plate2,H12; 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°