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Performance of a highly scalable and extraction-free RNA-seq method

ABSTRACT: RNA-seq workflows have become progressively more efficient over time however, RNA extraction still remains a significant bottleneck. For small numbers of sample, highly efficient RNA extraction is simple to perform but becomes costly and laborious at the scale of hundreds of samples. Our prior work has demonstrated that qPCR can be accurately performed using bulk cells samples lysate instead of RNA extraction. We combined this method with the recently developed simple method for rapid RNA-seq library prep; Smart-3SEQ (Foley et al., 2019) and hypothesized that bulk RNA-seq can be performed in a multi-well plate format, and at low cost by performing RNA-seq library prep cDNA synthesis using in-lysate RNA followed by Smart-3SEQ. The result shows success of our approach in various levels: 1) all quality control measures were achieved, 2) Gene expression profiles, gene differential expression and the response patterns reported by in-lysate and purified RNA library highly correlate with each other, and 3) in-lysate RNA-seq library prep performed similar to the gold standard used here (Illumina Truseq) for DEG calling.

ORGANISM(S): Homo sapiens

PROVIDER: GSE164650 | GEO | 2021/01/22

REPOSITORIES: GEO

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Project description:Abstract: Wallerian degeneration (WD) is a process of autonomous distal degeneration of axons upon injury. Macrophages (MP) of the peripheral nervous system (PNS) are main cellular agent controlling this process. Some evidences suggest that resident PNS-MP along with MP of hematogenous origin may be involved but whether these two subsets exert distinct functions is unknown. Combining MP-designed fluorescent reporters mice, and coherent anti-stoke raman scattering (CARS) imaging of the sciatic nerve, we deciphered the spatio-temporal choreography of resident and recently recruited MP after injury and unveiled distinct functions of these subsets with recruited MP responsible of efficient myelin stripping and clearance while resident MP were involved in axonal regrowth. This work provides clues to tackle selectively cellular processes involved in neurodegenerative diseases. Methods: (relevant for this GEO dataset): RNAseq of sciatic nerve macrophages from mice after CCI: Sorted cells were lysed in 100µl of RA1/TCEP buffer (NucleoSpin RNA XS, Macherey-Nagel), snap frozen in liquid nitrogen and stored at -80C until RNA extraction. All samples were processed in parallel and RNA extraction (without Carrier RNA but with on-column DNase treatment) was performed according to manufacturer's instructions (NucleoSpin RNA XS, Macherey-Nagel). RNA was eluted in RNAse-free water. Preparation of cDNA libraries for RNAseq was done using the SmartSeq method according to manufacturer's instructions (SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing, Clontech/TaKaRa). Due to the low number of cells, the total amount of eluted RNA was used as starting material for reverse transcription, followed by 18 cycles of pre-amplification. 1ng of cDNA were used for RNAseq sequencing library preparation, according to manufacturer's instructions (Nextera XT DNA Library Preparation, Illumina). Final samples pooled library prep were sequenced on a Nextseq 500 ILLUMINA with MidOutPut cartridge (2x130Millions of 75 bases reads) with 2 runs (4plex and 5plex), corresponding to 2x30Millions of (paired-end) reads per sample after demultiplexing.

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Performance of a highly scalable and extraction-free RNA-seq method

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