|
| Status |
Public on Sep 04, 2018 |
| Title |
lna_gene_jurkat_cd3d_resampled_lna |
| Sample type |
SRA |
| |
|
| Source name |
Jurkat cell line
|
| Organism |
Homo sapiens |
| Characteristics |
sample type: Jurkat cell line
|
| Extracted molecule |
total RNA |
| Extraction protocol |
10x 5' end Illumina libraries at 2nM concentration were diluted 20 fold and PCR amplified with Truseq Pcr F and R oligo for 14 cycles with 1 unit of Phusion. PCR results were confirmed by gel electrophoresis and the remaining sample was Ampure XP purified at a ratio of 1.8x bead/pcr volume. Samples were eluted in 10µl and molarity was determined using a Qubit. 10µl of 100nM library was mixed with 1 µl of 10µM DNA molecule in anneallng buffer (10mM Tris 8.0, 50mM NaCl) and heated to 98˚ for 10 minutes followed by slow cooling to 59˚-64˚ depending on the LNA and held O/N at the annealing temperature. Annealed DNA was added in equal volume to washed Dynabead M270 streptavidin beads at the annealing temperature. Beads were incubated for 15 minutes with gentle shaking. Beads were washed 5x in bind and wash buffer at the annealing temperature. Beads were eluted in 20 µl of elution solution (50mM NaCl, .1mM EDTA) heated for 10 minutes at 100˚ and centrifuged briefly to pellet the streptavidin-bound LNA molecules which inhibit PCR, this elution was done 2 times to ensure all the DNA was removed from the LNA. 10µl of streptavidin purified input DNA was added to PCR using Truseq Pcr F and R for 8 cycles. PCR products were ampure purified, confirmed on the Tapestation D1000, and quantified by the Qubit and submitted for sequencing. Read 1 positions 1-16 contain the 16bp cell barcode, and positions 17-26 contain a 10bp UMI. The remainder of read 1 contains the 5' of the cDNA. Read 2 also contains the cDNA.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Description |
Resampled single cell RNA-seq library from the 10x genomics 5' end library prep. Jurkat cells were isolated and an LNA probe was used to hybridize CD3D mRNA for targeted resequencing.
lna_gene_jurkat_bcs.txt
|
| Data processing |
Data analysis pipeline and custom scripts are provided in a github repository (https://github.com/rnabioco/scrna-subsets). Single cell RNA-Seq libraries were preprocessed to append the read 1 sequence to the paired read 2 read id (append_read_name) followed by quality trimming and poly(A) tail removal from read 2 using cutadapt (v. 1.8.3 -a 'A{{100}}' -O 6 -m 20 -q 10) (Martin 2011). Reads were next aligned with STAR (Dobin et al. 2013) to either the human genome assembly (hg38) for the PBMC experiments or a genome with both human (hg38) and mouse (mm38) sequences for all other experiments. Sequence headers in the human/mouse combined genome were prefixed with either an “H_” or “M_” to designate human or mouse references respectively. Following alignment, BAM files were processed to extract the cell barcode and UMI sequences into tags (CN and BX) within the BAM file (barcode_tag_bam). The cell barcode was error corrected against a list of cell barcodes, either as known well barcodes (Wafergen experiments), or generated from the original 10x genomics single cell libraries processed with the 10x genomics software cellranger (v. 2.1.1). Cell barcodes within edit distance of 1 of the known barcodes were considered valid cell barcodes and corrected to the known barcode. Alignments not designated as multi-mapping that overlapped distinct exonic features were tagged in the BAM file (subread v. 1.6.0) (Liao et al. 2014). Gencode v25 annotations was used for human data, and a union reference containing Gencode v25 and mouse v11 was used for human/mouse datasets. UMIs per gene were enumerated per cell using umi-tools (v 0.5.3) (Smith et al. 2017) using the directional method to disambiguate similar UMI sequences.
Genome_build: mm38, hg38
Supplementary_files_format_and_content: Text files containing cell barcode sequences, count matrices of UMIs per gene per cell are provided. For the TCR single cell libraries JSON files are provided with detailed annotations for each assembled contig per cell.
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| |
|
| Submission date |
Sep 04, 2018 |
| Last update date |
Sep 05, 2018 |
| Contact name |
Kent Augustus Riemondy |
| E-mail(s) |
kent.riemondy@cuanschutz.edu
|
| Organization name |
University of Colorado School of Medicine
|
| Department |
Biochemistry and Molecular Genetics
|
| Street address |
12801 E 17th Ave
|
| City |
Aurora |
| State/province |
Colorado |
| ZIP/Postal code |
80045 |
| Country |
USA |
| |
|
| Platform ID |
GPL24676 |
| Series (1) |
| GSE119428 |
Recovery and analysis of transcriptome subsets from pooled single-cell RNA-seq libraries |
|
| Relations |
| BioSample |
SAMN09951386 |
| SRA |
SRX4637906 |
