dsRNA-mediated RNAi is performed as described above. Single cells in the control and NSL1 KD samples were carefully diluted, re-suspended and filtered to obtain single cell suspension. The cells are then captured using the 10x genomics Chromium Single Cell 3' Library and Gel Bead Kit v2, 4 rxns (10x Genomics, 120267) according to the manufacturer's instructions (Single Cell 3' Reagent Kits v2 User Guide, CG00052). Sample was loaded to a different lane on a chip (Chromium Single Cell A Chip Kit, 16 rxns, 1000009). After generation of nanoliter-scale Gel bead-in-EMulsions (GEMs), RNA contained in the GEMs were reverse transcribed in a Thermal Cycler (Bio Rad) ( 53°C for 45 min, 85°C for 5 min, hold at 4°C). Then, the single-strand cDNA was isolated and amplified. Finally, the amplified cDNA was fragmented, end-repaired, A-tailed and indexed using the Chromium i7 Multiplex Kit 96 rxns (120262). The clean-up steps during the library generation was performed with Ampure beads. These libraries were sequenced using the Illumina HiSeq platform with a 75-bp paired-end kit. Alignment and initial processing of sequencing data were performed with CellRanger package using the default settings. The package was used to demultiplex the data, generate FASTQ files, align the sequencing reads to the dm3 reference genome, filter cell and UMI (unique molecular identifier) barcodes.
GEO Accession
GSM3344630