Sequencing of mice kidneys (ONT, IsoSeq, Kinnex, Illumina)
Sequencing of kidneys dissected from five male C57BL6/J wild-type mice (3 months old). Kidney tissues were homogenized using FastPrep and total RNA extracted using the 16 LEV simplyRNA Purification Kit for Maxwell. cDNA synthesis and sample barcoding were performed with the NEBNext Single Cell/Low Input cDNA synthesis amplification kit (IsoSeq). Five IsoSeq libraries (K31–K35) were prepared using the IsoSeq SMRTbell prep kit 3.0 and sequenced on a PacBio Sequel IIe. For nanopore samples, cDNA synthesis and sample barcoding were performed using the Nanopore SQK-PCB111-24 PCR-cDNA Barcoding Kit, following the standard protocol and generating 8 libraries per sample. The 8 libraries were then mixed and split into 8 aliquots. Eight rounds of sequencing were conducted on R9 flow cells (FLO-PRO002) using a Nanopore PromethION 2. For PacBio Kinnex libraries, RNA from 3 mouse kidneys was used for library preparation using Kinnex full-length RNA kit and sequenced on a Revio at Pacific Biosciences. Finally, directional cDNA synthesis and sample barcoding were performed using Illumina TruSeq Stranded mRNA Kit, following the standard protocol. After pooling the libraries, the samples were sequenced at 150x2 bp on an Illumina NovaSeq at Novogene.
Sequencing of kidneys dissected from five male C57BL6/J wild-type mice (3 months old). Kidney tissues were homogenized using FastPrep and total RNA extracted using the 16 LEV simplyRNA Purification Kit for Maxwell. cDNA synthesis and sample barcoding were performed with the NEBNext Single Cell/Low Input cDNA synthesis amplification kit (IsoSeq). Five IsoSeq libraries (K31–K35) were prepared using the IsoSeq SMRTbell prep kit 3.0 and sequenced on a PacBio Sequel IIe. For nanopore samples, cDNA synthesis and sample barcoding were performed using the Nanopore SQK-PCB111-24 PCR-cDNA Barcoding Kit, following the standard protocol and generating 8 libraries per sample. The 8 libraries were then mixed and split into 8 aliquots. Eight rounds of sequencing were conducted on R9 flow cells (FLO-PRO002) using a Nanopore PromethION 2. For PacBio Kinnex libraries, RNA from 3 mouse kidneys was used for library preparation using Kinnex full-length RNA kit and sequenced on a Revio at Pacific Biosciences. Finally, directional cDNA synthesis and sample barcoding were performed using Illumina TruSeq Stranded mRNA Kit, following the standard protocol. After pooling the libraries, the samples were sequenced at 150x2 bp on an Illumina NovaSeq at Novogene.
Institute for Integrative Systems Biology, Spanish National Research Council