> For the complete documentation index, see [llms.txt](https://lulab1.gitbook.io/training/llms.txt). Markdown versions of documentation pages are available by appending `.md` to page URLs; this page is available as [Markdown](https://lulab1.gitbook.io/training/archive/archive-2019/part-iii.-case-studies/case-study-1.exrna-seq.md).

# Case Study 1. exRNA-seq

## Basic analyses for exRNA-seq

### a) Background Introduction

* [PPT](/training/archive/archive-2019/getting-startted.md#learning-materials): 0. Introduction of exRNA-seq.pdf (view on-line only, not downloadable)
* [Teaching Video](/training/archive/archive-2019/getting-startted.md#learning-materials): Week V - Part II. 0. Intr to exRNA-seq.mov

### b) Understand your data

**Type of RNA-seq**

* **Default:** (whole cell poly-A) RNA-seq (>200nt)
* **Other types:**
  * small RNA-seq (<50nt)
  * total RNA-seq (ribosome removed) (>200nt)
  * nonpolyA RNA-seq (ribosome removed) (>200nt)
* [**Different cell localizations**](/training/archive/archive-2019/part-iv.-appendix/appendix-ii.public-data.md#local)**:**
  * nuc. (total)
  * chromosome (total)
  * cyto. (poly-A)
* **Single cell RNA-seq**
* **exRNA-seq**
  * cell free/MV/exosome/RNP
  * small/long

**So what RNA-seq we are mapping and analyzing?**

* Sequencing machine ?
* Single-strand V.S. Paired-end ?
* Strand specific ?
* Size selection ?
* Poly-A enriched or total (ribosome removed) ?
* Cellular localization ?

### c) Organize your data

[**Data format**](https://genome.ucsc.edu/FAQ/FAQformat.html)**:**

* fasta
* fastaq
* gff/gtf
* bam
* bed
* bigwig

**Shared dirs/data:**

```
cd  #go to my home
ln -s /BioII/lulab_b/shared/genomes  .   #shared reference genome sequeunces and annotations
ln -s /BioII/lulab_b/shared/shared_scripts  .  #shared scripts in Lu Lab
ln -s /BioII/lulab_b/shared/projects/exRNA  shared_exRNA_projects   #shared projects' files
```

* \~/genomes/human\_hg38
  * sequences/ #sequences of reference genome (fasta format)
  * index/ # indexed genome sequences
  * gtf/ #annotation of reference genome

**Make your own project dir:**

```bash
cd # go to my home
vim .bashrc # see my example in /home/john/.bashrc (and /home/john/shortcuts)
mkdir github

mkdir -p ~/projects/exRNA
cd ~/projects/exRNA

alias mkpr="mkdir -p {data/mapped,scripts,analysis}"  # you can put this in your ~/.bashrc
mkpr
```

### d) Get the software ready

[Install bioinformatics software in Linux (centos)](https://github.com/lulab/training/wiki/cnode)

## Video

[@Youtube](https://youtu.be/ovSVpqcDB8o)

[@Bilibili](https://player.bilibili.com/player.html?aid=30591172\&cid=53394108\&page=1)

## Other pipelines for RNA-seq analyses

{% hint style="success" %}
We also recommend some other Tutorials/Pipelines you can learn from:

* **RNA-seq analysis** pipeline:: <https://github.com/mgonzalezporta/TeachingMaterial>
* **lncRNA analysis** pipeline: <http://webhome.weizmann.ac.il/home/igoru/PLAR/>
* **ENCODE** pipelines: <https://www.encodeproject.org/pipelines/>
  {% endhint %}