Deciding if a study used Bulk or single-cell RNA sequencing using the GEO data

Question

[JACKY]

I am reading several studies in GEO and having difficulty determining whether bulk RNA sequencing or single cell RNA sequencing was performed. I try to identify key clues like the type of normalization approach used or sequencing kits mentioned, but the distinction is not always clear to me (mainly I click on one of the samples and use what is written there). I would greatly appreciate any help in reviewing a few of these studies to assess if bulk or single cell RNA sequencing was used:

GSE125667, GSE99531, GSE118383, GSE98368, GSE125483

Thank you!

Answer 1

[ATpoint]

• GSE125667

Smart-seq is written in the methods text. That is single-cell. Every fastq file (pair) is a cell.

• GSE99531

High molecular weight RNA (> 200 bp) was extracted from 2.000–40.000 cells

Bulk RNA-seq as they extracted from many cells.

• GSE118383

Targeted RNA-seq libraries were built...

That's bulk as well, but targeted RNA-seq.

• GSE98368

Bulk, no mention of single-cell.

• GSE125483

As above.

---

Please see the method sections provided at GEO for submissions. They tell what the data are.

[JACKY]

So whenever I see Smart-seq, it's single-cell ? Also, I only need the relevant approach for the samples mentioned in the GEO page. So refering to the papers might be confusing sometimes, cuase they might mention stuff that are not relevant to the samples in GEO. For examples some papers may use both bulk and single-cell, but the provided samples in GEO would only be one of them.

[Ram]

Are you sure Smart Seq is single cell? I think it's fewer cells than bulk but not single cell and analysis wise is closer to bulk than sc.

[GenoMax]

There are kits for single cell called SMARTseq from Takara: https://www.takarabio.com/products/next-generation-sequencing/rna-seq/single-cell-rna-seq/smart-seq-mrna-single-cell-lp-and-smart-seq-mrna-single-cell

[JACKY]

I think Smart-seq in used in both bulk and single cell. So it's tuff here to identify which one is it ..

[ATpoint]

Read the methods. It's single-cell as suggested.

https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSM3580020

Single cell cDNA libraries were using the SMARTSeq v2 protocol (Picelli et al., 2014) with the following modifications: 1. Use of 10 µM TSO; 2. Use of 250 pg cDNA with 1/5 reaction of Illumina Nextera XT kit (Illumina, San Diego, CA, USA). All samples were subjected to an indexed paired-end sequencing run of 2x51 cycles on an Illumina HiSeq 2000 system (Illumina, San Diego, CA, USA) (188 samples/lane).

[dsull]

Smart seq is single cell, but you are correct that analysis wise is closer to bulk (unlike 10X, it doesn't have UMIs; also unlike 10X; it's not only sequencing from the 3' end of reads; and also has greater depth per cell than 10X). Smart seq also uses plates rather than droplets (so all the droplet QCing sometimes done for 10X single-cell might not apply to smart-seq).

[i.sudbery]

SMART-seq is a protocol for doing RNA-seq from very low amounts of input RNA. It is most commonly used for single-cell RNA-seq, but can also be used other sorts of low input sequencing (e.g. extra-cellular RNA, tissue slices etc).

Answer 2

[i.sudbery]

Single Cell RNA-seq is relatively new, expensive, and difficult. If a study doesn't say that it is single-cell, I would assume that it is bulk.

[dsull]

Agreed; look at both the GEO accession info and the methods of the paper. If it doesn't say single cell/nucleus, then it's bulk. When doing only "bulk" RNAseq in a study, people generally don't say "bulk RNAseq", they just say "RNAseq".