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Sample GSM4736301

Query DataSets for GSM4736301

Status

Public on Dec 31, 2020

Title

NCC-RbC-51 Xenograft, rep1

Sample type

SRA

Source name

NCC-RbC-51

Organism

Homo sapiens

Characteristics

tissue: Xenograft
condition: Xenograft

Treatment protocol

Normal retina, retinoblastoma, xenografts, cultured cell lines in standard plate and graphene sponge were used to perform RNA-seq.

Growth protocol

Retinoblastoma cell line NCC-RbC-51 (Riken, Japan) was maintained in Roswell Park Memorial Institute media (RPMI 1640, Gibco, A10491-01). These were the cells grown in 2 dimensions on normal tissue culture plate. The cells grown on graphene sponge were considered as invitro 3-dimensional model for cell growth. PLL was added onto the graphene foam surface in 1:10 (v/v) dilution with sterile water and incubated for 3 h prior to NCC-RbC-51 cell seeding followed by washes with PBS and allowed to dry. Prior to cell seeding on to the scaffold the cells were made into cancer cell clusters as single cell suspensions did not attach well on the foams and they went through the pores and attached to the bottom of the cell culture plate. The NCC-RbC-51 cells under normal culture conditions form grape like clusters of 50-100 cells. These clusters were retained during cell seeding by gentle collection of the cells with minimal media volume (~200μl for 1 cm2 foam). After incubation time, complete media were supplemented. Caution was taken during media supplementation as sudden jerky movements caused dislodging of the cells from the scaffold. The cells were grown for 72h and taken for global transcriptomic studies by RNA sequencing as tissue culture plate grown cells exhibited increased cell death after this time period.

Extracted molecule

total RNA

Extraction protocol

RNA extraction for sequencing experiments were performed using RNeasy Mini Kit (Qiagen, Cat# 74104) then quantified using Qubit RNA Assay HS (Invitrogen, Cat# Q32855). RNA was isolated from NCC-RbC-51 cells grown on tissue culture plate, graphene sponges and xenograft tissue s(N=3). RNA purity was checked using QIAxpert and RNA integrity was assessed on TapeStation using RNA ScreenTapes (Agilent, Cat# 5067-5576).
NEB Ultra RNA-Seq Library Prep kit protocol (NEB, E7530L) was used to prepare libraries for total RNA sequencing. RNA (200ng) was depleted ribosomal RNA (rRNA) using biotinylated, target-specific oligos combined with Ribo-Cop rRNA removal beads. Following purification, the ribodepleted RNA was fragmented using divalent cations under elevated temperatures. The cleaved RNA fragments were copied into first strand cDNA using reverse transcriptase. Second strand cDNA synthesis was performed, using DNA Polymerase I and RNase H enzyme. The cDNA fragments were then subjected to a series of enzymatic steps which repair the ends, tails the 3’ end with a single ‘A’ base, followed by ligation of the adapters. The adapter ligated products were then purified and enriched using the following thermal conditions: initial denaturation 98°C for 30sec; 13 cycles of - 98°C for 10sec, 65°C for 75sec; final extension of 65°C for 5mins. PCR products are then purified and checked for fragment size distribution on TapeStation using D1000 DNA ScreenTapes (Agilent, Cat# 5067-5582). The adapter sequences used are presented in Supplementary Table 3. Prepared libraries were quantified using Qubit High Sensitivity Assay (Invitrogen, Cat# Q32854). The obtained libraries were pooled and diluted to final optimal loading concentration before cluster amplification on Illumina flow cell. Once the cluster generation is completed, the cluster flow cell is loaded on Illumina HiSeq 4000 instrument to generate 40M, 100bp paired end reads. The raw reads were filtered using Trimmomatic for Quality scores and adapters. Filtered reads were aligned to human genome using splice aware aligner like HISAT2 to quantify reads mapped to each transcript.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 4000

Description

sailaja-2020-cells-expr.txt
Xeno_1

Data processing

RNASeq data was processed using kallisto (version 0.45.0) and human genome GRCh38 Ensembl version 94 annotation (Homo_sapiens.GRCh38.94.chr_patch_hapl_scaff.gtf). Transcript level TPM values were added together to generate a gene-level TPM value. A custom perl script was used to organize and normalize data using log2(TPM+1).
Genome_build: Genome assembly: GRCh38.p12, Gene annotatiopn: Homo_sapiens.GRCh38.94.chr_patch_hapl_scaff.gtf
Supplementary_files_format_and_content: tab-delimited text file includes log2(TPM+1) values for each Sample

Submission date

Aug 21, 2020

Last update date

Dec 31, 2020

Contact name

Debashis Sahoo

E-mail(s)

dsahoo@ucsd.edu

Phone

6508624736

Organization name

UCSD