Single-Cell Transcriptome Profiling by CEL-Seq2

The sequencing libraries were prepared using CEL-Seq2 (Hashimshony et al., 2016 (link)). RNA from sorted cells was extracted using TRIzol reagent (ThermoFisher, 15596018). 10 ng RNA was used for first strand cDNA synthesis using barcoded primers (the specific primers for each sample were listed in Supplementary Table S9). The second strand was synthesized by NEBNext Second Strand Synthesis Module (NEB, E6111L). The pooled dsDNA was purified with AMPure XP beads (Beckman Coulter, A63880), and subjected to in vitro transcription (IVT) using HiScribe T7 High Yield RNA Synthesis Kit (NEB, E2040S), then treated with ExoSAP-IT (Affymetrix, 78200). IVT RNA was fragmented using RNA fragmentation reagents (Ambion), and underwent another reverse transcription step using random hexamer RT primer-5’-GCC TTG GCA CCC GAG AAT TCC ANN NNN N-3’ to incorporate the second adapter. The final library was amplified with indexed primers: RP1 and RPI1 (Supplementary Table S9), and the bead purified library was quantified with 4200 TapeStation (Agilent Technologies), and paired-end sequenced on Nextseq 500 V2 (Illumina), Read 1: 15 cycles; index 1: 6 cycles; Read 2: 60 cycles.

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Silverstein N.J., Wang Y., Manickas-Hill Z., Carbone C., Dauphin A., Boribong B.P., Loiselle M., Davis J., Leonard M.M., Kuri-Cervantes L., Meyer N.J., Betts M.R., Li J.Z., Walker B., Yu X.G., Yonker L.M, & Luban J. (2021). Innate lymphoid cells and disease tolerance in SARS-CoV-2 infection. medRxiv.

Publication Preprint 2021

TRIzol reagent NEBNext Second Strand Synthesis Module AMPure XP beads HiScribe T7 High Yield RNA Synthesis Kit ExoSAP-IT RNA fragmentation reagents 4200 TapeStation Nextseq 500 V2

Cdna Cells Dsdna Library Primers Reverse transcription Synthesis Transcription Trizol

Corresponding Organization : Broad Institute

Other organizations : Ragon Institute of MGH, MIT and Harvard, Massachusetts General Hospital, Harvard University, University of Pennsylvania, Brigham and Women's Hospital, Massachusetts Institute of Technology, Howard Hughes Medical Institute

Top 5 similar protocols

Variable analysis

independent variables

Use of CEL-Seq2 protocol for library preparation
Use of TRIzol reagent for RNA extraction
Amount of RNA used for cDNA synthesis (10 ng)
Use of barcoded primers for cDNA synthesis
Use of NEBNext Second Strand Synthesis Module for second strand synthesis
Use of in vitro transcription (IVT) for RNA synthesis
Use of ExoSAP-IT treatment
Use of RNA fragmentation reagents
Use of random hexamer RT primer for second adapter incorporation
Use of indexed primers (RP1 and RPI1) for library amplification

dependent variables

Sequencing of the final library using Nextseq 500 V2 (Illumina)

control variables

Keeping the experimental conditions and procedures consistent across all samples

controls

Positive control: None mentioned
Negative control: None mentioned

Annotations

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