GSM8005396: Sample_C_FRE; Homo sapiens; RNA-Seq
Fresh or thawed cryopreserved CSF mixed with 10x 5' v2 RT Reagent Master Mix were immediately processed with the 10x Genomics Chromium Next GEM Single Cell 5' v2 kit (10x Genomics, Pleasanton, CA), cDNA amplified, and library constructed per the manufacturer's protocol. Library quality control (QC) was based on Agilent Tapestation 4200 HS D1000 screentapes (Agilent Technologies, Waldbronn, Germany). Multiplexed library pool QC was based on Agilent Tapestation 4200 HS D1000 and Kapa Library Quantification Kit for Illumina platforms (Kapa BioSystems, Boston, MA) and sequenced at shallow depths on Illumina's iSeq 100 v2 flow cell for 26x10x10x90 cycles for estimated reads per cell. After demultiplexing,  libraries were rebalanced based on reads per cell. Normalized pool QC was based on Agilent Tapestation 4200 HS D1000 and Kapa Library Quantification Kit for Illumina platforms and high depth sequenced on Illumina's NovaSeq 6000 S4 v1.5 flow cell for 26x10x10x90 cycles, to obtain a sequencing depth of 50K reads/cell (whole transcriptome libraries). Reads were aligned to reference 2020-A (Ensembl 98) transcriptome and quantified using Cell Ranger v6.0.2 multipipeline. Fresh or thawed cryopreserved CSF mixed with 10x 5' v2 RT Reagent Master Mix were immediately processed with the 10x Genomics Chromium Next GEM Single Cell 5' v2 kit (10x Genomics, Pleasanton, CA), cDNA amplified, and library constructed per the manufacturer's protocol. Library quality control (QC) was based on Agilent Tapestation 4200 HS D1000 screentapes (Agilent Technologies, Waldbronn, Germany). Multiplexed library pool QC was based on Agilent Tapestation 4200 HS D1000 and Kapa Library Quantification Kit for Illumina platforms (Kapa BioSystems, Boston, MA) and sequenced at shallow depths on Illumina's iSeq 100 v2 flow cell for 26x10x10x90 cycles for estimated reads per cell. After demultiplexing,  libraries were rebalanced based on reads per cell. Normalized pool QC was based on Agilent Tapestation 4200 HS D1000 and Kapa Library Quantification Kit for Illumina platforms and high depth sequenced on Illumina's NovaSeq 6000 S4 v1.5 flow cell for 26x10x10x90 cycles, to obtain a sequencing depth of 50K reads/cell (whole transcriptome libraries). Reads were aligned to reference 2020-A (Ensembl 98) transcriptome and quantified using Cell Ranger v6.0.2 multipipeline. Fresh or thawed cryopreserved CSF mixed with 10x 5' v2 RT Reagent Master Mix were immediately processed with the 10x Genomics Chromium Next GEM Single Cell 5' v2 kit (10x Genomics, Pleasanton, CA), cDNA amplified, and library constructed per the manufacturer's protocol. Library quality control (QC) was based on Agilent Tapestation 4200 HS D1000 screentapes (Agilent Technologies, Waldbronn, Germany). Multiplexed library pool QC was based on Agilent Tapestation 4200 HS D1000 and Kapa Library Quantification Kit for Illumina platforms (Kapa BioSystems, Boston, MA) and sequenced at shallow depths on Illumina's iSeq 100 v2 flow cell for 26x10x10x90 cycles for estimated reads per cell. After demultiplexing,  libraries were rebalanced based on reads per cell. Normalized pool QC was based on Agilent Tapestation 4200 HS D1000 and Kapa Library Quantification Kit for Illumina platforms and high depth sequenced on Illumina's NovaSeq 6000 S4 v1.5 flow cell for 26x10x10x90 cycles, to obtain a sequencing depth of 50K reads/cell (whole transcriptome libraries). Reads were aligned to reference 2020-A (Ensembl 98) transcriptome and quantified using Cell Ranger v6.0.2 multipipeline.