input

Instructions to obtain processed data

From terminal (recommended):

wget https://ftp.ncbi.nlm.nih.gov/geo/series/GSE225nnn/GSE225599/suppl/GSE225599_bcell.rds.gz #b cells
wget https://ftp.ncbi.nlm.nih.gov/geo/series/GSE225nnn/GSE225599/suppl/GSE225599_cytotoxic.rds.gz #cd8 t, nk, dn t
wget https://ftp.ncbi.nlm.nih.gov/geo/series/GSE225nnn/GSE225599/suppl/GSE225599_final_dataSet_H.rds.gz #just healthy
wget https://ftp.ncbi.nlm.nih.gov/geo/series/GSE225nnn/GSE225599/suppl/GSE225599_final_dataSet_HvO.rds.gz #combined
wget https://ftp.ncbi.nlm.nih.gov/geo/series/GSE225nnn/GSE225599/suppl/GSE225599_helper.rds.gz #cd4 t
wget https://ftp.ncbi.nlm.nih.gov/geo/series/GSE225nnn/GSE225599/suppl/GSE225599_myeloid.rds.gz #myeloid cells

gunzip *.rds.gz

From R (NOTE: be sure to change dest to the path you want to download to!):

utils::download.file("https://ftp.ncbi.nlm.nih.gov/geo/series/GSE225nnn/GSE225599/suppl/GSE225599_bcell.rds.gz", dest = "/pwd/to/dir/bcell.rds.gz")
utils::download.file("https://ftp.ncbi.nlm.nih.gov/geo/series/GSE225nnn/GSE225599/suppl/GSE225599_cytotoxic.rds.gz", dest = "/pwd/to/dir/cytotoxic.rds.gz")
utils::download.file("https://ftp.ncbi.nlm.nih.gov/geo/series/GSE225nnn/GSE225599/suppl/GSE225599_final_dataSet_H.rds.gz", dest = "/pwd/to/dir/final_dataSet_H.rds.gz")
utils::download.file("https://ftp.ncbi.nlm.nih.gov/geo/series/GSE225nnn/GSE225599/suppl/GSE225599_final_dataSet_HvO.rds.gz", dest = "/pwd/to/dir/final_dataSet_HvO.rds.gz")
utils::download.file("https://ftp.ncbi.nlm.nih.gov/geo/series/GSE225nnn/GSE225599/suppl/GSE225599_helper.rds.gz", dest = "/pwd/to/dir/helper.rds.gz")
utils::download.file("https://ftp.ncbi.nlm.nih.gov/geo/series/GSE225nnn/GSE225599/suppl/GSE225599_myeloid.rds.gz", dest = "/pwd/to/dir/myeloid.rds.gz")

You can then use the R function GEOquery::gunzip, terminal gunzip, or prefered method to unzip the files

Instructions to obtain count matrices from NCBI

Navigate to directory in which you wish to download the data and run the following:

wget https://ftp.ncbi.nlm.nih.gov/geo/series/GSE225nnn/GSE225599/suppl/GSE225599_RAW.tar

(Alternatively, you can download the zip folder by visiting the GSE225599 page)

Then upacked with:

tar -xf GSE225599_RAW.tar

(Or, right click and extract all)

To rearrange the file structure for easy loading into Seurat, follow the code chunks below (Alternatively, manually rearrange):

First, get the provided deCoder.tsv file in the directory you wish to house the input files then:

touch rearrange.sh

Then, copy the contents of the below script into the rearrange.sh file

#!/usr/bin/env bash

while read line
do
    old=$(echo "$line" | cut -f1)
    new=$(echo "$line" | cut -f2)
    
    mkdir $new
    
    barcodes="./${old}_barcodes.tsv.gz"
    feats="./${old}_features.tsv.gz"
    mtx="./${old}_matrix.mtx.gz"

    mv $barcodes ./$new/$new\_barcodes.tsv.gz
    mv $feats ./$new/$new\_features.tsv.gz
    mv $mtx ./$new/$new\_matrix.mtx.gz

done < deCoder.tsv

After transfering the code, run:

bash rearrange.sh

From there you can follow the runScript_PBMC_analysis.R script

Instructions to obtain raw data from SRA

Navigate to directory of interest and run for each file you wish to pull down. Then with SRA toolkit installed run:

NOTE: all raw data files are just under 2TB of data

prefetch -v --max-size=55000000 SRR23525324 #smallest file for ex
fastq-dump --gzip --split-files SRR23525324

From there you will have to modify the file names to be compatible with the cellranger input (if using cellranger). It expects [Sample Name]_S1_L00[Lane Number]_[Read Type]_001.fastq.gz -- feel free to reach out if you have trouble