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Sample GSM1683228

Query DataSets for GSM1683228

Status

Public on May 21, 2015

Title

PS041-2_R2_0_289

Sample type

SRA

Source name

U87 human glioma cells

Organism

Homo sapiens

Characteristics

cell type: U87 human glioma cells

Growth protocol

Cells were cultured in DMEM supplemented with 10% FBS.

Extracted molecule

polyA RNA

Extraction protocol

RNA was extracted from individual cells in individual microfluidic chambers following cell lysis by Triton X-100 and freeze-thaw.
mRNA from individual cells was reverse transcribed with a primer containing a cell-identifying barcode followed by oligo(dT). Following second strand synthesis using DNA Polymerase I and reagents from the MessageAmp II kit (Ambion), ds-cDNA from all barcoded individual cells was pre-amplified by in vitro transcription using T7 RNA polymerase in a pool. The pools of amplified RNA from each lane of the microfluidic device were individually reverse transcribed using barcoded random hexamers containing both a unique molecular identifier (random 8-base barcode) followed by a lane-identifying barcode (6-base barcode). Illumina adapters were inserted on either end of the library during the two previous reverse transcription steps and were used to then enrich the library by PCR. The pooled library was sequenced on an Illumina NextSeq 500.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NextSeq 500

Description

PS041_0
3'-end RNA-Seq
processed data file: PS041-2_R2_0.txt

Data processing

Read 1 of our single cell RNA-Seq data contains a cell-identifying barcode sequence followed by poly(dT), and read 2 contains a 8-base UMI followed by a 6-base lane-identifying barcode and a transcript sequence. We first demultiplex the reads based on the lane-identifying barcode while recording the corresponding UMI using a custom Python script. We then map the remainder of read 2 to the human genome and transcriptome (hg19, Ensembl annotation from Illumina iGenomes) using the STAR aligner. Mapped reads for each lane are then demultiplexed based on the cell-identifying barcodes in read 1 and assigned to a gene using HTSeq. Both the lane- and cell-identifying barcodes were allowed to have a single-base mismatch during demultiplexing. We note that >30% of reads map to the PhiX spike-in genome from the Illumina sequencing kit.
We collected the set of reads that uniquely mapped to the transcriptome and assigned an address comprised of its cell-identifying barcode, gene, UMI, and mapping position. We then filtered the reads to identify unique molecules. Reads with identical addresses were collapsed to a single molecule. In addition, reads with identical cell-identifying barcodes, genes, mapping positions, and with UMIs having a Hamming distance less than or equal to two were collapsed to a single molecule. Finally, because the mapping positions produced by STAR do not necessarily correspond to the beginning of a read, we further considered reads to originate from identical molecules if they had identical genes, cell-identifying barcodes, UMIs with a Hamming distance less than or equal to two, and a mapping position within five bases.
To identify barcodes that correspond to actual individual cells in our device, we filtered the observed cell-identifying barcodes by progressively downsampling the corresponding gene profiles to the same number of total reads and assessing the number of unique molecules detected from each cell-identifying barcode. After excluding cell-identifying barcodes having zero associated molecules, we found the distribution of associated unique molecules to be bimodal, with one small subpopulation having nearly as many unique molecules as reads at low read totals. We found the size of this subpopulation to be in excellent agreement with our device imaging data. We took these 598 profiles to represent the actual individual cells captured in our device with a barcoded bead.
We conducted more detailed analysis on 396 single-cell profiles with the highest coverage in our data set across all five lanes of the microfluidic device for PS034 and 247 single-cell profiles from PS041. Raw fastq data from read 2 of those 396 cells from PS034 and 247 cells from PS041 are provided here. Note that the UMI for each read appears in the comment line of each fastq entry.
The processed data files contain the number of molecules counted for each gene based on counting reads with HTSeq and filtering the UMIs to identify unique molecules. If two UMIs had a Hamming distance less than three, they were considered to be the same UMI. If two reads with identical UMIs mapped to the transcriptome to within 6 bases of each other, they were considered identical molecules. In addition, any molecules considered to be identical by the above defintion, but that appeared in two different cells were eliminated to mitigate any sources of cross-talk resulting from PCR recombination
Genome_build: hg19
Supplementary_files_format_and_content: The processed data files contain the number of molecules counted for each gene based on counting reads with HTSeq and filtering the UMIs to identify unique molecules.

Submission date

May 13, 2015

Last update date

May 15, 2019

Contact name

Peter A Sims

E-mail(s)

pas2182@columbia.edu

Organization name

Columbia University

Street address

3960 Broadway, Lasker 203AC