title: GSM4403138: single_cell_barcoded_SmartSeq2 [14-2]; Homo sapiens; RNA-Seq
library_construction_protocol: Microglia were isolated by mechanical tissue dissociation and Percoll gradient centrifugation at 4°C followed by flow cytometry of viable (DAPI-, DRAQ5+), CD45+, CD11B+ cells. All 25 RNA samples, with RIN values varying between 5.7 and 9.9, were enriched for poly(A)+ messenger RNA using NEXTflex Poly(A) Beads (BIOO Scientific, #NOVA-512980) according to the protocol in the manual. Fourteen µL of this mRNA-enriched poly(A)-tailed RNA was used as the input for the NEXTflex Rapid Directional qRNA-Seq kit (Bioo Scientific, #NOVA-5130-04). Library preparation was performed according to the manufacturers protocol. Quality and concentration of libraries from individual samples were assessed using the High Sensitivity dsDNA kit (Agilent, 067-4626) on a 2100 Bioanalyzer (Agilent) and a Qubit 2.0 Fluorometer (Life Technologies). Subsequently, individual libraries were combined into 2 sequencing pools of 13 samples each with equal molar input. 75bp paired-end sequencing was performed on an Illumina NextSeq 500 system. PhiX was added at 5% to both pools as an internal control before sequencing. single cell RNA-Seq, bc-Smart-seq2 :Microglia were isolated by mechanical tissue dissociation and Percoll gradient centrifugation at 4°C followed by flow cytometry of single, viable (DAPI-, DRAQ5+), CD45+, CD11B+ cells. single cell RNA-Seq, bc-Smart-seq2 : After cell lysis and barcoded poly-dT primer annealing, RNA was reverse-transcribed with a biotinylated barcoded template switching oligo (BC-TSO). Primer-dimers and small fragments were removed by exonuclease treatment. cDNA libraries were amplified and multiplexed. When cDNA libraries showed an average size of 1.5-2 kb, libraries were tagmented and indexed according to the Illumina Nextera XT DNA preparation kit (Illumina, FC-131-1024) with an input of 500 pg cDNA. Samples were sequence on an Ilumina NextSeq 500. using 75 bp paired-end reads. Read 1 comprises the poolbarcode and read 2 the cDNA. 10X Genomics: Microglia were isolated by mechanical tissue dissociation and Percoll gradient centrifugation at 4°C followed by flow cytometry of viable (DAPI-, DRAQ5+), CD45+, CD11B+ cells. 10X Genomics: The single-cell barcoded libraries were constructed according to the instructions of the Single Cell 3' Reagent Kits v2 (10x Genomics). Briefly, cells were loaded into a slot of a Chromium chip and GEMs were incubated in a thermal cycler to generate barcoded cDNA. After amplification, the cDNA was fragmented and processed for sequencing by ligating adapters and sample indices. The libraries were sequenced on an Illumina NextSeq 500 system with a sequencing depth up to 20,000 reads per cell.
attributes.tag: GEO Accession
attributes.value: GSM4403138