Materials & Methods

Acknowledgements

Please mention Single Cell Discoveries in your acknowledgements if we helped you with single-cell sequencing experiments and/or data analysis. We always love to know when our clients have published data generated by our services, so please also let us know if your paper will come online, so we can help promote it. Sample text for acknowledgements:

“We thank Single Cell Discoveries for their help with project design, single-cell sequencing services, and data analysis”

Thank you!

SORT-seq

Viable single cells were FACS sorted into 384-well plates, called cell capture plates, that 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 nl 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 (Muraro et al. 2016 with primers described in van den Brink et al. 2017). 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.3   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 1×75 bp Illumina kit (read 1: 26 cycles, index read: 6 cycles, read 2: 60 cycles)  

10x Genomics

Single-cell mRNA sequencing was performed at Single Cell Discoveries, a single-cell sequencing service provider in the Netherlands, according to standard 10x Genomics [5’ v2 Single Cell Immune Profiling, or 3’ V3.1 Single Cell Gene Expression] dual index chemistry guidelines.  

Choose your applicable preservation method, for example:  

  • Cryopreservation: samples were cryopreserved following [X]protocol and thawed according to 10x genomics guidelines. Cells were washed, filtered, and counted. Viability was assessed and was [e.g., >70% or >90%]. 
  • Methanol fixation: samples were methanol fixed according to 10x Genomics demonstrated protocol “Methanol Fixation of Cells for Single Cell RNA Sequencing” (CG000136) and frozen at -80°C for [X] weeks. Prior to loading the cells on the 10x Chromium chip, cells were rehydrated according to the same demonstrated protocol. Cells were filtered and counted. Fresh: samples were collected and immediately proceeded to 10x Genomics processing. Cells were filtered and counted. Viability was assessed and was [e.g., >70% or >90%]. 

Cells were loaded in a concentration of 700-1200 cells/µl on a 10x Genomics chip, and the chip was run on a Chromium controller to initiate GEM formation. Library preparation was performed, following standard 10x Genomics guidelines [select one of the following options: 5’ v2 (incl. or excl. BCR/TCR) = CG000331 guide, 3’ v3.1 = CG000315 guide, 5’ v2 with feature barcoding (incl. or excl. BCR/TCR) = CG000330 guide , 3’ v3.1 with feature barcoding = CG000317 guide], to generate Gene Expression (GEX) libraries. [optional: In addition TCR/BCR/feature barcoding libraries were generated]. The resulting DNA libraries were paired-end sequenced on an [Illumina Novaseq S1, or Illumina Novaseq S2, or Illumina Novaseq S4] with an Illumina 2×150 bp kit (read 1: 28 cycles, index reads: 2×10 cycles, read 2: 90 cycles) , to obtain an average of [X] reads per cell for the GEX library [optional: and an average of [X] reads per cell for the TCR/BCR/feature barcoding library].   

Bulk RNA sequencing

Please contact us since it can differ between projects.

Updated on March 31, 2022

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