We used SORT-seq to sequence the transcriptome from single cells and store FACS information from single cells (index files) (Muraro et al., 2016 (link)). All sorts were carried out using BD FACSJazz or BD FACSInflux. Unless mentioned otherwise, we used the following protocol for both model systems mentioned in this study. We lysed cells by incubating them at 65°C for 5 min, and then used Nanodrop II liquid handling platform (GC biotech) to dispense RT and second strand mixes. The aqueous phase was separated from the oil phase after pooling all cells into one library, followed by IVT transcription. The CEL-Seq2 protocol was used for library prep (Muraro et al., 2016 (link)). Primers consisted of a 24 bp polyT stretch, a 4 or 6bp random molecular barcode (UMI), a cell-specific 8bp barcode, the 5′ Illumina TruSeq small RNA kit adaptor and a T7 promoter. We used TruSeq small RNA primers (Illumina) for preparation of Illumina sequencing libraries and then paired-end sequenced them at 75 bp read length using Illumina NextSeq at approximately 45 million and 30 million reads for zebrafish kidney marrow and human pancreatic libraries respectively.
Truseq small rna primers
Manufactured by Illumina
Sourced in Netherlands, United States
The TruSeq small RNA primers are a set of oligonucleotide sequences designed for the library preparation and sequencing of small RNA molecules, such as microRNAs, small interfering RNAs, and Piwi-interacting RNAs. The primers are used in the Illumina TruSeq small RNA library preparation workflow to enable the amplification and adapter ligation of small RNA targets prior to sequencing.
Automatically generated - may contain errors
Lab products found in correlation
Nextseq, by Illumina (7 mentions)
Truseq small rna kit adapter, by Illumina (5 mentions)
Nextseq 500 platform, by Illumina (4 mentions)
Nextseq 500, by Illumina (3 mentions)
Nextseq platform, by Illumina (3 mentions)
384 well hardshell plates, by Bio-Rad (2 mentions)
Ercc spike ins, by Agilent Technologies (2 mentions)
Dntps, by Promega (2 mentions)
Mineral oil, by Merck Group (2 mentions)
Ambion purelink rna mini kit, by Thermo Fisher Scientific (2 mentions)
Facsjazz, by BD (1 mentions)
Facs influx, by BD (1 mentions)
Nextera xt, by Illumina (1 mentions)
Hiseq 2500, by Illumina (1 mentions)
Bioanalyzer, by Agilent Technologies (1 mentions)
Vapour lock, by Qiagen (1 mentions)
Facsaria fusion cell sorter, by BD (1 mentions)
Filcon, by BD (1 mentions)
Nextseq 500 high output, by Illumina (1 mentions)
M8410, by Merck Group (1 mentions)
20 protocols using truseq small rna primers
1
Single-Cell RNA-Seq with SORT-seq and CEL-Seq2
2
Single-Cell RNA-Seq Library Preparation Protocols
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Library preparation for the Pgc+ cells was performed by the Single-Cell Genomics Core Facility of the WT Sanger Institute. Briefly, mRNAs isolated from single cells from each condition were amplified using the SMARTSeq2 protocol (Picelli et al., 2014 (link)). Multiplexed sequencing libraries were prepared from amplified cDNA using Nextera XT (Illumina) and sequenced on a HiSeq 2500 running in rapid mode.
Library preparation and sequencing of the Gif lineage cells were performed by Single Cell Discoveries. Single-cell RNA sequencing was performed according to an adapted version of the SORT-seq protocol (Muraro et al., 2016 (link)) with primers described in another paper (van den Brink et al., 2017 (link)). Cells were heat-lysed at 65 °C followed by cDNA synthesis. After second-strand cDNA synthesis, all the barcoded material from one plate was pooled into one library and amplified using in vitro transcription (IVT). Following amplification, library preparation was done following the CEL-Seq2 protocol (Hashimshony et al., 2016 (link)). To prepare a cDNA library for sequencing, TruSeq small RNA primers (Illumina) were used. The DNA library was paired-end sequenced on an Illumina Nextseq™ 500, high output, with a 1x75 bp Illumina kit (read 1: 26 cycles, index read: 6 cycles, read 2: 60 cycles).
Library preparation and sequencing of the Gif lineage cells were performed by Single Cell Discoveries. Single-cell RNA sequencing was performed according to an adapted version of the SORT-seq protocol (Muraro et al., 2016 (link)) with primers described in another paper (van den Brink et al., 2017 (link)). Cells were heat-lysed at 65 °C followed by cDNA synthesis. After second-strand cDNA synthesis, all the barcoded material from one plate was pooled into one library and amplified using in vitro transcription (IVT). Following amplification, library preparation was done following the CEL-Seq2 protocol (Hashimshony et al., 2016 (link)). To prepare a cDNA library for sequencing, TruSeq small RNA primers (Illumina) were used. The DNA library was paired-end sequenced on an Illumina Nextseq™ 500, high output, with a 1x75 bp Illumina kit (read 1: 26 cycles, index read: 6 cycles, read 2: 60 cycles).
3
SORT-seq Partially Automated scRNA-seq
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scRNA-seq libraries were generated using the SORT-seq protocol (54 (link)), a partially robotized method based on the CEL-Seq2 protocol (55 (link)). Briefly, single cells were lysed at 65°C for 5 min, and second-strand mixers and reverse transcriptase were then added to the wells using the Nanodrop II liquid handling platform (GC Biotech). After reverse-transcribing the mRNA of each cell, double-stranded complementary DNAs (cDNAs) from single cells were pooled, and in vitro transcription was performed for linear amplification, which resulted in amplified RNA. TruSeq small RNA primers (Illumina) were used to prepare the Illumina sequencing libraries, and these DNA libraries were then sequenced paired-end at 75–base pair read length using the Illumina NextSeq (performed commercially by Single Cell Discoveries, Utrecht, The Netherlands). Both the RNA yield of the amplified RNA and the quality and concentration of the final cDNA libraries were assessed by Bioanalyzer (Agilent).
4
Single-cell Transcriptomics of Zebrafish Cells
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Single-cell sequencing libraries were prepared using SORT-seq (Muraro et al., 2016 (link)). Live cells were sorted into 384-well plates with Vapor-Lock oil containing a droplet with barcoded primers, spike-in RNA and dNTPs, followed by heat-induced cell lysis and cDNA syntheses using a robotic liquid handler. Primers consisted of a 24 bp polyT stretch, a 4 bp random molecular barcode (UMI), a cell-specific 8 bp barcode, the 5′ Illumina TruSeq small RNA kit adapter and a T7 promoter. After cell lysis for 5 min at 65°C, RT and second strand mixes were distributed with the Nanodrop II liquid handling platform (Inovadyne). After pooling all cells in one library, the aqueous phase was separated from the oil phase, followed by IVT transcription. The CEL-Seq2 protocol was used for library prep (Hashimshony et al., 2016 (link)). Illumina sequencing libraries were prepared with the TruSeq small RNA primers (Illumina) and paired-end sequenced at 75 bp read length on the Illumina NextSeq platform. Mapping was performed against the zebrafish reference assembly version 9 (Zv9).
5
Single-cell RNA-seq of Regulatory T Cells
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Live CD3+CD4+CD25+CD127low cells were sorted from fresh SF (Supplementary figure 1a ) into 384‐well hard shell plates (Bio‐Rad, Hercules, USA) with 5 μL of vapour‐lock (QIAGEN, Hilden, Germany) containing 100–200 nL of RT primers, dNTPs and synthetic mRNA Spike‐Ins and immediately spun down and frozen to −80°C. Cells were prepared for SORT‐seq as previously described.49 Illumina sequencing libraries were then prepared with the TruSeq small RNA primers (Illumina, San Diego, USA) and sequenced single‐end at 75 basepair read length with 60 000 reads per cell on a NextSeq500 platform (Illumina). Sequencing reads were mapped against the reference human genome (GRCh38) with BWA.
6
Single-cell sequencing of pancreatic progenitors
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Cells differentiated at day 13 were harvested with TrypLE Select as described above, resuspended in Stage 5 medium supplemented with 10 μM Y-27632, filtered using a 50 μM sterile Filcon (BD Biosciences) and sorted using a FACSAria Fusion cell sorter (BD) directly into 2 x 384-well plates with ERCC spike-ins (Agilent), reverse transcription primers and dNTPs (both Promega), according to the gating shown in Figure S10 B. Repartition was as follow: 216 cells for each NKX6-1-GFP+, NEUROG3-mCherry+ and NKX6-1-GFP+/NEUROG3-mCherry+ population and 104 cells for the negative population. Single cell sequencing was performed according to the Sort-seq method68 (link) by Single Cell Discoveries. Briefly, Sequencing libraries were generated with TruSeq small RNA primers (Illumina) and sequenced paired-end at 60 and 26 bp read length, respectively, on the Illumina NextSeq. Reads were mapped to the human GRCh38 genome assembly. Sort-seq read counts were filtered to exclude reads with identical library-, cell- and molecule barcodes. UMI counts were adjusted using Poisson counting statistics.68 (link)
7
Single-cell RNA sequencing protocol
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The cell capture plates were ordered from Single Cell Discoveries, a single-cell sequencing service provider based in the Netherlands. Each well of a cell capture plate contains a small 50 µl droplet of barcoded primers and 10 μl of mineral oil (Sigma M8410). After sorting, plates were immediately spun and placed on dry ice. Plates were stored at -80° C. Plates were shipped on dry ice to Single Cell Discoveries, where single-cell RNA sequencing was performed according to an adapted version of the SORT-seq protocol (38 (link)) with primers described in van den Brink et al., 2017 (39 (link)). Cells were heat-lysed at 65° C followed by cDNA synthesis. After second-strand cDNA synthesis, all the barcoded material from one plate was pooled into one library and amplified using in vitro transcription (IVT). Following amplification, library preparation was done following the CEL-Seq2 protocol (40 (link)) to prepare a cDNA library for sequencing using TruSeq small RNA primers (Illumina). The DNA library was paired-end sequenced on an Illumina Nextseq™ 500, high output, with a 1x75 bp Illumina kit (read 1: 26 cycles, index read: 6 cycles, read 2: 60 cycles).
8
Single-cell transcriptomics using SORT-seq
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Single-cell mRNA sequencing was performed according to an adapted version of the SORT-seq protocol (26 (link)), using barcoded poly-A primers described by van den Brink and colleagues (27 (link)). In short, single cells were FACS sorted into 384-well plates containing 384 different poly-A barcoded primers and Mineral oil (Sigma). After sorting, plates were snap-frozen on dry ice and stored at −80°C. Subsequently, cells were heat-lysed at 65°C followed by cDNA synthesis using the CEL-Seq2 protocol (28 (link)) using robotic liquid handling platforms Nanodrop II (GC Biotech) and Mosquito (TTP Labtech). After second strand cDNA synthesis of poly-A transcripts, the barcoded material was pooled into libraries of 384 cells and amplified using in vitro transcription (28 (link)). Following amplification, the rest of the CEL-seq2 protocol was followed for preparation of the amplified cDNA library, using TruSeq small RNA primers (Illumina) as previously described (26 (link)). The DNA library was paired-end sequenced on an Illumina Nextseq500, high output, 1 × 75 bp.
9
Plasmid DNA Extraction and Sequencing
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Plasmid DNA from each collected population was prepared according to Medina-Cucurella & Whitehead64 (link) using Zymoprep Yeast Plasmid Miniprep kits in either individual Eppendorf tubes (D2004) or 96-well plate format (D2007) and plasmid DNA was eluted in 30 μL nuclease free water. 15 μL of eluted plasmid DNA was further purified with exonuclease I and lambda exonuclease. The barcode region of the purified DNA was amplified using 25 PCR cycles with Illumina TruSeq small RNA primers following Kowalsky et al ‘Method B’67 . Amplicons were sequenced on either an Illumina MiSeq (4A8/CC12.1/COV2–2489 sort) or NovaSeq6000 (S1/HA sorts) by Rush University with single end reads.
10
Single-Cell RNA-Seq Library Preparation
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A modified version of Cel-seq2 was used to generate single cell cDNA libraries. Briefly, the sorted 384-well plates were defrosted on ice and the cells were lysed and the primers annealed at 65 °C. Primers contained a 24 bp polyT stretch, a 6 bp unique molecular identifier (UMI), a cell-specific 8 bp barcode, the 5′ Illumina adaptor and a T7 promoter. Reverse transcription, second strand synthesis and in vitro transcription were performed according to the Cel-seq268 (link) with the exception of the cDNA cleanup step prior to IVT, whereby the volume of the Agencourt AMPure XP beads (A63880, Beckman Coulter) was 5% of the total volume of beads used in the original protocol. Following IVT, the aRNA was fragmented, cleaned up and reverse transcribed with a random hexamer primer containing the sequence complimentary to the 3’ Illumina adaptor. Fragments containing both adaptors were selected for by PCR using TruSeq Small RNA primers (Illumina) and cleaned up using a 0.7X bead to cDNA ratio. The quality and concentration of the final cDNA library was checked using a High Sensitivity Bioanalyzer (Agilent). Libraries were pooled at equimolar ratios and sequenced on the Illumina Nextseq.
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