GSM5068216: Myeloid scRNA-seq; Mus musculus; RNA-Seq
RNA from snap frozen tissue biopsies was extracted using the RNA easy kit (Qiagen, Hilden, Germany). RNA quality was assessed using the Fragment Analyzer (Advances Analytical Technologies Inc., Ankeny, USA). RNA-seq libraries were prepared from 250 ng of total RNA. The RNA samples were depleted for ribosomal RNAs with the Illumina TruSeq Stranded Total RNA Gold kit. Ribosomal-RNA depleted RNA was then converted in cDNA and amplified with the kit Nugen Ovation® RNA-Seq System V2. It was used to generate sequencing libraries with the Illumina TruSeq DNA Nano kit. A Chromium Next GEM Chip G (10X genomics) was loaded with the appropriate number of cells, and the sequencing libraries prepared with the Chromium Next GEM Single Cell V(D)J Reagent Kits v1.1 following the manufacturer's recommendations. Briefly, an emulsion encapsulating single cells, reverse transcription reagents and cell barcoding oligonucleotides was generated. After the actual reverse transcription step, the emulsion was broken, and double stranded cDNA generated and amplified in a bulk reaction. For 5' gene expression library this cDNA was fragmented, ligated to a sequencing adaptor, and PCR amplified. For V(D)J library preparation, a similar approach was followed except that 2 steps of PCR based V(D)J target enrichment were performed prior to fragmentation. Libraries were quantified by a fluorimetric method and their quality assessed on a Fragment Analyzer (Agilent Technologies). Cluster generation was performed with 140 to 165 pM of an equimolar pool from the resulting libraries using the Illumina HiSeq 3000/4000 PE Cluster Kit reagents. Sequencing was performed on the Illumina HiSeq 4000 using HiSeq 3000/4000 SBS Kit reagents according to 10X Genomics recommendations (26 cycles read1, 8 cycles i7 index read, and 91 cycles read2).