single cell RNA-seq of Homo sapiens: adult brain middle temporal gyrus: Sample H18.03.011.13.001
"We performed single cell RNA-seq on human virus-infected neurons by 1 hour digestion at 30oC in carbogenated ACSF.1/trehalose + blockers + papain, followed by gentle trituration in Low-BSA Quench buffer, shallow spin gradient centrifugation (100g 10 minutes at room temperature) into High-BSA Quench buffer, and resuspension into Cell Resuspension Buffer. We also employed Myelin Bead Removal Kit II (Miltenyi catalog # 130-096-733) at 1/20 to remove myelin debris, and PE-anti CD9 clone eBioSN4 (Thermo Fisher catalog # 12-0098-42) at 1/50 to sort away contaminating glial cells. Then we sorted single SYFP2+ labeled human neurons for sequencing, one cell per well, using SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing (Takara, 634894) to reverse transcribe poly(A) RNA and amplify full-length cDNA according to the manufacturer's instructions. We performed reverse transcription and cDNA amplification for 18 PCR cycles in 8-well strips, in sets of 12-24 strips at a time. All samples proceeded through Nextera XT DNA Library Preparation (Illumina FC-131-1096) using Nextera XT Index Kit V2 Set A (FC-131-2001). Nextera XT DNA Library prep was performed according to manufacturer's instructions except that the volumes of all reagents including cDNA input were decreased to 0.4x or 0.5x by volume.  Then sample libraries were pooled evenly and sequenced with paired-end 50bp reads on NextSeq machines (SeqMatic, Fremont CA USA)."