SAMN32519300
External Id
University of California, San Francisco
INSDC center name
2022-12-31T00:00:00Z
INSDC first public
2022-12-31T12:15:20.600Z
INSDC last update
SRS16328734
INSDC secondary accession
live
INSDC status
Human
NCBI submission model
Human.1.0
NCBI submission package
SRS16328734
SRA accession
56
age
GRCh38
assembly
Raleigh Lab UCSF Helen Diller Family Comprehensive Cancer Center 1450 3rd Street, San Francisco 94218
biomaterial_provider
sgRNA library was packaged into lentivirus using HEK-293T cells. HEI-193 cultures were transduced to an MOI of 0.1 and FACS sorted for sgRNA+ cells after 48 hours. Radiotherapy was then delivered to either 0Gy or 1.8Gy x 5 daily fractions using the X-Rad 320 irradiator (Precision X-Ray). Following completion of radiotherapy, cells were harvested with Trypsin and prepared following the Nuclei Isolation for Single Cell Multiome ATAC + Gene Expression Sequencing 10x Protocol (10x Genomics, CG000365 Rev C). Briefly, 1-2 105 cells were incubated in chilled Lysis Buffer (Tris-HCl (pH 7.4) 10 mM, NaCl 10 mM, MgCl2 3 mM, Tween-20 0.1%, Nonidet P40 Substitute 0.1%, Digitonin 0.01%, BSA 1%, DTT 1mM, RNase inhibitor 1 U/µL) on ice for 4 minutes, washed with Wash Buffer (Tris-HCl (pH 7.4) 10 mM, NaCl 10 mM, MgCl2 3 mM, Tween-20 0.1%, BSA 1%, DTT 1 mM, RNase inhibitor 1 U/uL) 3 times, and resuspended in diluted nuclei buffer. Nuclei were counted and membrane integrity was evaluated by Trypan staining on the Countess II FL Automated Cell Counter (Thermo). Nuclei suspensions were diluted to approximately 3,000 nuclei/µL and processed according to the 10x Chromium Next GEM Single Cell Multiome ATAC + Gene Expression protocol (CG000338 Rev A). To recover sgRNA identities from single-nuclei RNA fractions, CROP-seq guides were amplified into dual-indexed Illumina libraries from the cDNA product of the 10x multiome protocol as described above with a three-round hemi-nested PCR as previously described85. 15 ng of full-length cDNA product was amplified with primers binding to the sgRNA constant region and 10x Genomics Read 1 Adaptor. Two subsequent PCR steps were performed to introduce i5 and i7 indices and Illumina P5 and P7 adaptors. After the first round of PCR, each product was size-selected using SPRI beads at 1.0x. Subsequent PCRs were conducted with 1 ng product. The final PCR product was size selected with 0.5x and then 1.0x SPRI beads and library quality was assessed by TapeStation high-sensitivity D1000 analysis (Thermo). CROP-seq libraries were pooled with gene expression libraries and sequenced on an Illumina NovaSeq 6000 using the paired end 100 bp protocol.
design_description
HEI193_5x1_8_ATAC_2_S5_L001_R1_001.fastq.gz
filename
HEI193_5x1_8_ATAC_2_S5_L002_I1_001.fastq.gz
filename10
HEI193_5x1_8_ATAC_2_S5_L003_I1_001.fastq.gz
filename11
HEI193_5x1_8_ATAC_2_S5_L004_I1_001.fastq.gz
filename12
HEI193_5x1_8_ATAC_2_S5_L001_R3_001.fastq.gz
filename13
HEI193_5x1_8_ATAC_2_S5_L002_R3_001.fastq.gz
filename14
HEI193_5x1_8_ATAC_2_S5_L003_R3_001.fastq.gz
filename15
HEI193_5x1_8_ATAC_2_S5_L004_R3_001.fastq.gz
filename16
HEI193_5x1_8_ATAC_2_S5_L002_R1_001.fastq.gz
filename2
HEI193_5x1_8_ATAC_2_S5_L003_R1_001.fastq.gz
filename3
HEI193_5x1_8_ATAC_2_S5_L004_R1_001.fastq.gz
filename4
HEI193_5x1_8_ATAC_2_S5_L001_R2_001.fastq.gz
filename5
HEI193_5x1_8_ATAC_2_S5_L002_R2_001.fastq.gz
filename6
HEI193_5x1_8_ATAC_2_S5_L003_R2_001.fastq.gz
filename7
HEI193_5x1_8_ATAC_2_S5_L004_R2_001.fastq.gz
filename8
HEI193_5x1_8_ATAC_2_S5_L001_I1_001.fastq.gz
filename9
fastq
filetype
Illumina NovaSeq 6000
instrument_model
schwannoma
isolate
HEI193_5x1_8_ATAC_2_S5
library_ID
Homo sapiens
http://purl.obolibrary.org/obo/NCBITaxon_9606
organism
ILLUMINA
platform
male
sex
cells
tissue
snARC_HEI193_5x1_8Gy_ATAC_2
title