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Sample GSM2149386

Query DataSets for GSM2149386

Status

Public on Oct 17, 2016

Title

JM3_1

Sample type

SRA

Source name

Glioblastoma

Organism

Homo sapiens

Characteristics

cell type: GBM

Growth protocol

HEK293T and glioblastoma cells (U87, KS4, JM3, JM4) were mycoplasma tested. Naïve Stem Cell Culture: Human ESC line WA09 (female) (WiCell, Madison, WI) was transitioned from conventional primed conditions to 5iAF naïve state by closely following the conversion protocol described in Theunissen et al., 2014. Naïve hESCs were maintained and expanded in a medium containing 50:50 N2B27/Neurobasal (GIBCO) with 1mM glutamine (Invitrogen), 1% NEAA (Invitrogen), 0.1mM β-mercaptoethanol (Sigma), and freshly supplemented with 10 µM Y-27632 ROCKi (Stemgent), 1 µM PD0325901 MEKi (Sigma), 1 µM IM-12 GSK3i (Sigma), SB590885 BRAFi (Sigma), 1 µM WH-4-023 SRCi (Sigma), 20 ng/ml Activin A (R&D), 0.5 % KOSR (Invitrogen), 8 ng/ml bFGF (R&D), 50 ug/ml BSA (Sigma), and 20 ng/ml hLIF (Millipore) (Theunissen et al. 2014). Cells were propagated on irradiated E12.5 MEFs (GIBCO) seeded at 250 k cells/well of a 6 well (9.5 cm2), and once colonies reached confluence they were dissociated and expanded by single cell using Accutase (GIBCO). WA09 (H9) has been mycoplasma tested and validated for pluripotency by teratoma assay (in primed state) as well as immunocytochemistry (for both naive and primed). Based on our Smart-seq2 data, our cells exhibit a transcriptional profile that are indicative of the previously reported works that we aimed to reproduce. This cell line was obtained from WiCell Research Institute, Inc. from which we hold the MTA (license). Primed Stem Cell Culture: Conventional primed human hESC line WA09 (WiCell, Madison, WI) was maintained on a layer of mouse embryonic fibroblasts (MEF), in media consisting of: DMEM: F12 (Invitrogen), 20 % KSR (Invitrogen), 4 ng/ml bFGF (R&D), 2 mM glutamine (GIBCO), 0.1 mM non-essential amino acids (Invitrogen), 0.1 mM ß-Mercaptoethanol (Sigma). Once colonies reached confluence they were mechanically passaged through dissociation using a 26-gauge needle (VWR).

Extracted molecule

total RNA

Extraction protocol

Cells were lysed directly into the reaction tube. No RNA extraction was performed.
Plates were pre-deposited with 3 µl of lysis buffer (0.13 % Triton-X-100, 4 u recombinant RNase Inhibitor, Takara). After single cell sorting, the plates were transferred to -80 °C for long storage. After thawing the plate, 1 µl of 5.8 S rRNA masking oligo (5 pmol) was added and the whole plate was incubated at 72 °C for 20 minutes. Then, 2 µl of 3’ adaptor ligation reaction was added (20 pmol 3’ adaptor oligo, 8.33 % PEG 8000, 50 u T4 RNA Ligase 2, truncated KQ, NEB, 0.83 X T4 RNA ligase buffer, NEB, 4 u recombinant RNase Inhibitor, Takara) and reaction was incubated at 30 °C for 6 h followed by 4 °C for 10 hours. Next, 3 µl of RT primer and free adaptor removal enzymes were added (200 pmol RT primer, 2.5 u Lambda exonuclease, NEB, 10 u 5’ deadenylase, NEB) and the reaction was incubated at 30 °C for 15 minutes followed by 37 °C for 15 minutes. Next, 2 µl of 5’ adaptor ligation reaction was added (45 pmol 5’ adaptor oligo, 0.68 mM Tris-buffered ATP, Thermo Fisher, 4 u T4 RNA ligase, Thermo Fisher, 0.23 X T4 RNA ligase buffer, NEB ) and the mix was incubated at 37 °C for 1 hour. Reverse transcription reaction was performed by adding 7 µl of RT reaction (1.28 X Taq DNA Polymerase PCR Buffer, Thermo Fisher, 8.33 mM DTT, 0.42 mM/each dNTP, 4 u recombinant RNase Inhibitor, 150 u Superscript II reverse transcriptase, Thermo Fisher). The PCR amplification is carried out by adding 35 µl of the reagents (0.94 X Phusion HF buffer, 1 u Phusion Hot Start II DNA Polymerase, Thermo Fisher, 0.12 mM/each dNTP, 1.89 µM RP1 primer) and incubating at 98 °C for 30 seconds followed by 13 cycles of 98 °C for 10 seconds, 60 °C for 30 seconds and 72 °C for 30 seconds and a final incubation at 72 °C for 5 minutes. At last, 1 µl of the amplified product is transferred to a fresh tube and the second PCR reaction is added (2 µM indexed primers, 0.2 µM RP1 primer, 1 X Phusion HF buffer, 0.5 u Phusion Hot Start II DNA Polymerase, Thermo Fisher, 0.2 mM/each dNTP) and incubating at 98 °C for 30 seconds followed by 13 cycles of 98 °C for 10 seconds, 67 °C for 30 seconds and 72 °C for 30 seconds and a final incubation at 72 °C for 5 minutes. The amplified libraries of single cells are then pooled and purified with DNA Clean & Concentrator™-5, Zymo research. All samples have been sequenced 1x 43 bp or 1x 51 bp using Illumina HiSeq 2500 instrument.

Library strategy

ncRNA-Seq

Library source

transcriptomic

Library selection

size fractionation

Instrument model

Illumina HiSeq 2000

Description

allcells_mirna_postqc.txt

Data processing

Processing raw reads: The sequence in FastQ file corresponding to UMIs were removed from sequence reads and appended to the read name (for later analyses). Adapter and polyA sequences were removed from reads using cutadapt v1.8.1, with minimum overlap between adapter and the 3’ of the read set to 1 nt. Reads shorter than 18 nt after adaptor trimming were discarded.
Alignment: Trimmed reads were aligned to the human genome (hg38) using STAR v2.4.0 with parameters --outSAMstrandField intronMotif --outFilterMismatchNoverLmax 0.04 --outFilterMatchNmin 18 --outFilterScoreMinOverLread 0 --outFilterMatchNminOverLread 0 --alignIntronMax 1. We did not allow any mismatches within the first 25 nucleotides and allowed only 1 mismatch in the remaining part of the sequence reads (25 – 41 nts). Spliced alignments were disabled and hard/soft-clipping was disabled for the 5’ of the read. Soft-clipping of up to 3 nts were allowed at the 3’ end of the read in order to account for RNA editing and 3’ tRNA modifications such as addition of CCA. Reads mapping with insertions or deletions were removed.
Counting unqiue molecules: We collapsed PCR amplicons and counted RNA molecules using the adjacency network approach (dedup_umi.py at https://github.com/CGATOxford/UMI-tools), where reads with UMIs having a single Hamming distance from another UMI were collapsed. We separated RNA molecule counts into two categories based on the extent of the alignment of our sequenced read with the human genome. Reads aligning over the full sequence (41 nts) were assigned as precursor molecules, whereas reads aligning with 40 or less nts were assigned as a potential small RNA. Potential small RNAs were further scrutinized to find instances were trimmed 3’ ends matched the genomic sequence after the alignment (cases where actual RNA sequence matched adapter sequence, false positive adapter trimming). Consequently, these molecules were instead assigned to the precursor RNA.
Annotating molecules: We estimated expression for annotation transcripts in the following databases (Gencode V22, Mirbase V21 and GtRNAdb). During the quantification procedure, molecules were hierarchical assigned to annotated biotypes in the following order: Mirbase miRNAs, tRNAs from GtRNAdb, small RNA biotypes from Gencode (such as snoRNAs, snRNAs, rRNAs etc.), and lastly the remaining of Gencode transcripts such as protein coding and lincRNAs. We counted molecules from both intronic and exonic regions of the protein coding and lincRNAs in an attempt to capture small RNAs transcribed from these regions. We used a weighing approach to assign molecules of sequences mapping to multiple genomic location, by divided by number of annotated location the sequence aligned to. Finally, for miRNAs we collapsed molecules supporting the same miRNA that were expressed from different genomic loci (i.e. where the microRNAs gene was present in multiple genomic locations).
Filtering samples: We excluded libraries with fewer than one million reads (before alignment) and if the number of expressed transcripts were more than two standard deviations of the median number of expressed transcripts obtained within the cell population (to filter out potential doublets and low-quality libraries). All analyses in the manuscript was carried out on small RNA molecule counts (i.e. amplicon corrected values), except otherwise noted.
Genome_build: hg38
Supplementary_files_format_and_content: Tabular expression matrix

Submission date

May 10, 2016

Last update date

May 15, 2019

Contact name

Ilgar Abdullayev

E-mail(s)

[email protected]

Organization name

Karolinska Institutet

Department

Cell and Molecular Biology