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Sample GSM6177626 Query DataSets for GSM6177626
Status Public on Aug 05, 2022
Title Exp1 10x Genomics Gene Expression
Sample type SRA
Source name Single-cell RNA-Seq of mouse pancreatic adenocarcinoma in WT and Rag-_- mice
Organism Mus musculus
Characteristics cancer type: Pancreatic adenocarcinoma
organ: Pancreas
method: 10x Genomics Gene Expression
Growth protocol For mouse pancreatic adenocarcinoma, Rag1-_- and WT C57BL_6 mice were obtained from Jackson Laboratories (Bar Harbor, ME). The KrasG12D;Tp53R172H;Pdx1Cre (KPC) derived cell line FC1242 was utilized for orthotopic injection of 100,000 cells into the tail of pancreata of 8-12 week old C57BL_6 or Rag mice. To model liver and peritoneal metastases, mice received FC1242 via splenic (1,000,000 cells) and intraperitoneal (100,000 cells) injection, respectively. Tumors were harvested 2-3 weeks after injection.
Extracted molecule total RNA
Extraction protocol For inDrop single-cell RNA-Seq of patient tumor samples, cell suspension was generated from minced tumor tissue using human tumor dissociation kit (Miltenyi) prior to droplet encapsulation and individual barcoding of cells using the inDrop platform. Cell lysis was performed in droplets to release RNA, and reverse transcription and barcoding took place in droplets at 50C for 2h. For 10x Genomics spatial transcriptomics of patient tumor samples, 10 µm cryosections were mounted onto spatially barcoded arrays where reverse transcription of cellular transcripts took place in situ after tissue permeabilisation for 12 minutes. For 10x Genomics single-cell RNA-Seq of mouse tumors, cell suspension was generated from minced tumor tissue using the mouse tumor dissociation kit (Miltenyi) enzymes D and R prior to droplet encapsulation and individual barcoding of cells using the 10x Genomics platform.
For inDrop single-cell RNA-seq library construction, we followed the inDrop library construction protocol detailed by Klein et al, Cell 2015, with minor changes. Briefly, RNA was reverse transcribed (RT) with SuperScript III (Invitrogen) in droplets. Droplet emulsions were broken, and post-RT material underwent second strand syntehsis and in vitro transcription using the T7 High Yield Enzyme mix (New England BiolaBS). RNA was fragmented for 105 seconds with 1X fragmentation reagent (1CellBio) prior to RT with random hexamers, eliminating the need for an adaptor ligation step. Cycles required for final library amplification was assessed by quantitative PCR (qPCR) with KAPA HiFi Hot Start PCR Mix (KAPA Biosystems) and EvaGreen dye (Biotium). Final libraries were amplified with KAPA HiFi Hot Start PCR mix for 9 to 13 cycles. inDrop library size assessed on a DNA BioAnalyzer chip following the manufacturer's instructions (Agilent). For 10x Genomics spatial transcriptomic and single-cell RNA-Seq library construction, we followed the manufacturer's instructions.
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina NextSeq 500
Data processing Illumina RTA v2 software was used for basecalling and quality determination.
Raw sequencing data obtained from the inDrop scRNA-Seq method were processed using a custom-built pipeline, available online (https:__github.com_flo-compbio_singlecell). Briefly, the “W1” adapter sequence of the inDrop RT primer was located in the barcode read (the second read of each fragment), by comparing the 22-mer sequences starting at positions 9-12 of the read with the known W1 sequence (“GAGTGATTGCTTGTGACGCCTT”), allowing at most two mismatches. Reads for which the W1 sequence could not be located in this way were discarded. The start position of the W1 sequence was then used to infer the length of the first part of the inDrop cell barcode in each read, which can range from 8-11 bp, as well as the start position of the second part of the inDrop cell barcode, which always consists of 8 bp. Cell barcode sequences were mapped to the known list of 384 barcode sequences for each read, allowing at most one mismatch. The resulting barcode combination was used to identify the cell from which the fragment originated. Finally, the UMI sequence was extracted, and reads with low-confidence base calls for the six bases comprising the UMI sequence (minimum PHRED score less than 20) were discarded. The reads containing the mRNA sequence (the first read of each fragment) were mapped by STAR 2.5.1 with parameter “—outSAMmultNmax 1” and default settings otherwise36. Mapped reads were split according to their cell barcode and assigned to genes by testing for overlap with exons of protein-coding genes.
Raw sequencing data obtained from the 10x Genomics Visium method were processed using the SpaceRanger pipeline.
Raw sequencing data obtained from the 10x Gene Expression method were processed using the CellRanger pipeline.
Assembly: hg38, mm10
Supplementary files format and content: for inDrop single-cell RNA-Seq data: genes as rows and cells as columns. For 10x Genomics spatial transcriptomic data: output of SpaceRanger pipeline, including gene expression and spatial contents. For 10x Genomics single-cell RNA-Seq: output of CellRanger pipeline, including gene expression, features and barcodes.
Submission date May 23, 2022
Last update date Aug 06, 2022
Contact name Dalia Barkley
Organization name NYU Langone Health
Department Institute for Computational Medicine
Lab Yanai
Street address 435 E 30th st
City New York
State/province NY
ZIP/Postal code 10016
Country USA
Platform ID GPL19057
Series (1)
GSE203612 Cancer cell states recur across tumor types and form specific interactions with the tumor microenvironment
BioSample SAMN28600484
SRA SRX15419212

Supplementary file Size Download File type/resource
GSM6177626_Exp1_RagKO_barcodes.tsv.gz 49.7 Kb (ftp)(http) TSV
GSM6177626_Exp1_RagKO_features.tsv.gz 272.9 Kb (ftp)(http) TSV
GSM6177626_Exp1_RagKO_matrix.mtx.gz 59.2 Mb (ftp)(http) MTX
GSM6177626_Exp1_RagWT_barcodes.tsv.gz 49.7 Kb (ftp)(http) TSV
GSM6177626_Exp1_RagWT_features.tsv.gz 272.9 Kb (ftp)(http) TSV
GSM6177626_Exp1_RagWT_matrix.mtx.gz 55.7 Mb (ftp)(http) MTX
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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