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Sample GSM5325377

Query DataSets for GSM5325377

Status

Public on Jun 10, 2021

Title

L12_ROI23_Tumor

Sample type

SRA

Source name

NSCLC tumor

Organism

Homo sapiens

Characteristics

tissue: L12
aoi_type: Tumor
roi: ROI23

Extracted molecule

total RNA

Extraction protocol

Samples were incubated with DNA-oligo barcoded RNA-ISH probes which were conjugated with a UV-photocleavable linker following standard ISH protocols, along with flourescently labeled antibodies for visualization of morphological structures. Regions of interest within the tissue were illuminated with UV light and oligo barcodes were physically aspirated from the tissue and collected into microtiter plates by the GeoMx® Digital Spatial Profiler (DSP) platform. For more information about DSP protocols please see Merritt et al. Nature Biotech 2020 (doi: 10.1038/s41598-020-63539-x)
Each collection of oligo tags from one well (representing an AOI from the tissue section) was indexed with i7xi5 unique dual indexes using GeoMx SeqCode primers with 18 cycles of PCR. After PCR, indexed AOIs were pooled and purified in two rounds of AMPure XP PCR purification using 1.2x bead:sample ratio.

Library strategy

OTHER

Library source

transcriptomic

Library selection

other

Instrument model

NextSeq 550

Description

photocleaved oligonucleotide barcodes
Samples were pooled during library preparation and split across lanes. Individual FASTQ files were generated for each lane, forward & reverse primers. The Fastq files contain the same identifier as the sample name for aggregation

Data processing

Library strategy: GeoMx-Seq
Adapter trimming with trim galore, trimGaloreOpts = " --hardtrim5 26 --dont_gzip"
If paired-end, merge overlapping R1 and R2 with flash2, flash2Opts = " -m 26 -e 26 -f 26 -s 1 -r 27"
Extract UMIs in bowtie2, umiExtractOpts = " --bc-pattern=NNNNNNNNNNNNNN"
Align RTS_IDs (probe barcodes) using bowtie2, bowtie2Opts = " --end-to-end -L 4 --trim5 0 --trim3 0 --norc"
Deduplication using UMI-tools, umiDedupOpts = " --edit-distance-threshold=1"
Genome_build: N/A, sequencing of synthetic tags and alignment to whitelist of RTS_IDs (probe barcodes) found in the config.ini output from the GeoMx DSP platform
Supplementary_files_format_and_content: Digital Count Conversion (DCC) file format outputted from GeoMx NGS Pipeline, contains software versions, scan attributes, GeoMx NGS pipeline parameters and output metrics, Q30 scores, and list of deduplicated counts per RTS_ID (probe barcode)
Supplementary_files_format_and_content: Values represented in the collapsed target counts tab are the geometric mean of the probes for a given, removing any targets flagged as outliers. Analyzed counts represent the upper quartile normalized collapsed counts across the study after removing QC flagged segments.

Submission date

May 20, 2021

Last update date

Jun 10, 2021

Contact name

Patrick Danaher

Organization name

NanoString Technologies, Inc.

Street address

530 Fairview Ave N

City

Seattle