Guidelines for low-input RNA-seq (SmartSeq-2)

GENERAL GUIDELINES FOR SAMPLE PREPARATION

The SmartSeq-2 workflow is designed to sequence low amounts of RNA. It can be applied to single cells, a suspension of few cells (up to 300), or to low input RNA samples (40pg to 10ng total RNA). Below we provide details on how to prepare these types of samples.

CHECKLIST CRITICAL POINTS

Please check carefully the following aspects!

Single cells

For the single-cell approach individal cells should be sorted into 96 or 384-well plates with lysis buffer (recipe here) in each well (2µl/well if 96-well plate; 0.5µl/well if 384-well plate). After sorting, plates should be centrifuged shortly and stored at -80°C.

Notes on FACS sorting:

  • cell sorting should take place in a cooled system and it should not take longer than 1h. Long sorting times lead to decreased quality of the RNA.

  • a precise set up of cell sorting is critical, in order to ensure that the cell lands directly in the middle of the well – and thus directly into the lysis buffer. Wrong dispensing with the FACS instrument may lead to cells being stuck on the walls, resulting in RNA degradation.

Low cell numbers bulk mRNA Sequencing

For small bulk mRNA sequencing, cells (up to 300 cells/well) can be sorted directly into 8-strip PCR tubes pre-filled with 2µl lysis buffer (recipe here). Cell lysates must be stored at -80°C.

Low input bulk mRNA Sequencing

For low-input RNA samples, please collect cells in PBS and subsequently isolate the RNA (e.g. with Qiagen’s Micro RNeasy kit or Qiazol/Trizol alternatives for higher yields). Please elute RNA in 10µl nuclease-free water. (do not use DEPC-treated water.)

Given the low amount of RNA, checking the quality of the RNA is skipped. It is critical to use RNase inhibitors in the lysis buffer when working with cells. We generally have good experience with NEB Murine RNase Inhibitor (#M0314S), but for critical samples, other inhibitors might perform better (e.g. ThermoFisher RNaseOUT (#10777019) or Sigma Protector RNase inhibitor (#3335402001)).

SEQUENCING

Sequencing is limited to polyadenylated RNAs and not strand-specific. Standard sequencing for these workflows is paired-end 2 x 100bp. Different read lengths can be requested.

For single cells we recommend a sequencing depth of at least 1.5 mio read pairs/cell. For the cell bulk approach and low-input mRNA sequencing the coverage depends on the expected sample complexity, we recommend 20 - 40 mio read pairs / sample. We are glad to provide guidance with this.

In general, we recommend at least 3 biological replicates for a proper data analysis. Specificities of the species, sample, or project design, can influence the optimal sequencing coverage.

BIOINFORMATICS ANALYSIS

For the plate-based single-cell approach we apply our single-cell analysis pipeline, which generates the following information: number of cells that were sequenced, estimation of background noise, average number of genes expressed per cell|nuclei, clustering analysis, and a list of genes that characterizes each cluster.

For the cell suspension and low-input bulk RNA approaches, we apply the pipeline used for bulk RNA sequencing. This pipeline comprises all steps from initial quality control, read alignment and read count, correction for batch effects, sample correlation, cluster analyses (e.g. PCA), visualisations of variable and highly expressed genes, and differential gene expression analyses.

SAMPLE SUBMISSION

Preparing aliquots for sequencing

If needed, we can provide 8-strip tubes or plates for FACS sorting with pre-loaded lysis buffer. In this case, please contact us 1 week before cell sorting takes place.

Supported plastics for sample submission

Samples can only be submitted in 96-well plates, 384-well plates or 8-strip PCR tubes with single lids. Strips or plates that are fully compatible with our systems are Sarstedt strips (#72.991.002), Eppendorf 96-well twin.tec® skirted PCR plates (e.g. Cat.no. 0030128648) and 4titude 384-well FrameStar plates (Cat. no. 4ti-LB0384/C). These can be provided upon request. When submitting in plates please fill them column-wise w/o empty wells in between.

Please identify tubes/samples with clear and short labels, e.g. with the sample ID (created in Rosalind during online sample submission) or a running index clearly referenced under the corresponding samples submitted over the Rosalind Client Portal.

Plates should be labelled with LAB-Project ID, client name, plate number (when more than 1 plate), and sorting date. For submitted samples of the same batch we require that the input material doesn’t differ much between samples - i.e. roughly same number of cells or same amount of RNA for each sample.

Plates:

  • Eppendorf twin.tec® PCR Plate 96, skirted (Cat. no. 0030128648) or
  • 4titude FrameStar® 96 Well Skirted PCR Plate (Cat. no. 4ti-0960/RIG)
  • Eppendorf twin.tec®-PCR-Platte 384 LoBind (PN 0030129547)
  • 4titude 384-well plates (PN 4TI-0384-C)

PCR strips:

  • Sarsted PCR strip, 8 tubes with flat lid, Multiply® -µStrip (Cat no. 72.991.002)
  • VWR 8 strips (Cat no. 732-0545)

Providing project and sample information via the Rosalind Client Portal

Please log in to Rosalind to enter all necessary project information making use of the “Project Description” field during the project set-up dialogue. Give all relevant details about project design, sample preparation method and how the samples are eluted and/or preserved. Sample-specific information such as concentration, volume, used preparation protocols etc. should be provided when the samples are added to the created project.

Please also indicate if you require a bioinformatics analysis during project setup.

Submission of large sample batches

The Rosalind customer portal offers an Excel-based alternative for sample submission of more than 30 samples delivered in either 8 well strips, 96- or 384-well plates. You can specify these supported formats in the portal’s sample submission dialog and download a formatted Excel template for entering your sample details. Plate identifiers and well coordinates are pre-set to facilitate easy data entry. Once completed, please email the Excel file to genomecenter@tu-dresden.de. Do not upload individual samples to Rosalind in this case.

To submit samples, you must be registered in our Rosalind client portal. Therefore, please send an e-mail to genomecenter@tu-dresden.de with your contact details (name, working group, working group leader, your e-mail address and telephone number). This will enable us to set up an account for you. After you have received an automatic e-mail, please log in to Rosalind with your login details. If needed, we can reset your login details.

Delivering or shipping of samples

Once the project and sample information has been entered into the Rosalind Client Portal, please put the samples in a small plastic bag labeled with your name and Rosalind project ID.

Samples can be personally delivered to the lab at the (CRTD, 3rd floor south), daily from 9-12h and 13-16h. If needed, contact us to deliver samples outside of this time window (+49 351 458 82362 or email us at genomecenter@tu-dresden.de).

Please deliver samples under suitable conditions! When shipping your samples, please use an adequate-sized box and ensure a proper amount of cooling material to hold the sample temperature until it reaches us. The package should be addressed to:

DRESDEN-concept Genome Center
c/o Center for Regenerative Therapies Dresden
Technische Universität Dresden
Fetscherstraße 105
01307 Dresden

Following up project progress

You can accompany the status of your project in our Rosalind Client Portal. Documents with information about the preparation method and quality control of samples and libraries are provided for your project.

REFERENCES