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Sample GSM4060044

Query DataSets for GSM4060044

Status

Public on Sep 05, 2019

Title

coordinate reads for Jurkat cell experiment

Sample type

SRA

Source name

synthetic DNA

Organism

synthetic construct

Characteristics

cell line: n/a

Treatment protocol

Cells were resuspended. HEK293 and 3T3 cells were stained with Calcein Green and Red; Jurkat cells were stained with DRAQ5. Cells were then encapsulated in 80-um droplets.

Growth protocol

HEK293 and 3T3 cells were grown in DMEM with 10% FBS in a T75 flask. Jurkats were grown in RPMI-1640 with 10% FBS in a suspension culture.

Extracted molecule

total RNA

Extraction protocol

Cell-containing drops were merged with lysis buffer drops containing coordinate oligos and barcoded poly dT beads within microwells. Beads were then processed using the Drop-Seq protocol, using the modified CITE-seq/Cell hashing protocol to separate coordinate oligo reads from mRNA reads.
The coordinate library directly had sequencing adapters added with PCR. The mRNA library underwent tagmentation and sequencing adapter addition using the Illumina Nextera XT sample preparation kit. A custom Read 1 primer used in Drop-Seq was used during the sequencing of both libraries.
Drop-Seq; CITE-Seq/Cell hashing

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2500

Data processing

Basecalling was done with bcl2fastq v2.20
Bead barcode was extracted from position 1-12 of Read 1 and UMI was extracted from position 13-20 of Read 1.
For mRNA reads: Read2 was aligned to a reference human or mouse genome using STAR.
For coordinate reads: Read2 was aligned to a known list of well index sequences using custom Python code.
Digital gene expression matrices were created by, for each bead barcode group, counting the number of unique UMIs for each gene or well index sequence.
To link beads to nanowell position: UMI counts for each bead are scaled based on the number of total UMIs on the bead. Next, the off-target noise of each well index is estimated based on the average expression across all beads and subtracted from scaled UMI counts. The top x, y, and z well index captured on each bead is then extracted. Beads which the top well index is not at least 5 times as abundant as the next most abundant well index for any of the sets of x, y, and z well indexes are removed. The remaining beads are assigned to a nanowell position by matching the most abundant x, y, and z indexes on the bead to a lookup table of the expected x, y, and z positions at each nanowell position. Following position assignment, the bead barcodes of all beads matched at each nanowell position are collected. The columns on the gene expression matrix of all beads matched at the same nanowell position are merged, yielding a revised matrix where the columns represented nanowell positions instead of individual beads.
The gene expression matrix is then annotated by recorded cell fluorescence values obtained during printing.
Genome_build: hg18; mm10
Supplementary_files_format_and_content: Digital gene expression matricies by nanowell position (.csv)
Supplementary_files_format_and_content: Indexed flow cytometry data by nanowell position (.csv)

Submission date

Sep 04, 2019

Last update date

Jan 15, 2020

Contact name

Jesse Zhang

E-mail(s)

jesse.zhang@ucsf.edu

Organization name

UCSF

Department

BTS