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Sample GSM1444841 Query DataSets for GSM1444841
Status Public on Nov 24, 2014
Title L129_D05
Sample type SRA
 
Source name lumbar dorsal root ganglion
Organism Mus musculus
Characteristics cell picking session: A
cell picking temperature: RT
strain: C57/Bl6
gender: female
Growth protocol 6-8 week old C57/Bl6 mice
Extracted molecule total RNA
Extraction protocol Six lumbar DRGs (L4-L6) were dissected and dissociated using papain and collagenase/dispase followed by Percoll gradient centrifugation. The sample was always kept on ice apart from enzyme incubation. In some preparations a cooled microscope stage was used. The final cell pellet was suspended in 300 ml of L15 medium supplemented with B27. Cells were picked on an in house constructed picking setup built around an inverted Nikon microscope. Individual cells were sucked into a glass capillary attached to a CellTram syringe with visual control that individual cells were captured. The capillary tip was positioned in a well of a 96 well plate containing 3 microlitre of lysis buffer into which the cell was released by operating the CellTram syringe. 48 random cells were picked at each time within 40-60 minutes. The lysis buffer contained 20mMTris-HCl at pH8.0, 75 mM KCl, 6 mM MgCl2, 0.02% Tween-20 with 400 nM STRT-V3-T30 (5'-biotin-AAGCAGTGGTATCAACGCAGAGTCGACT(30)VN-3') and 400 nM STRT-V2-n (5'-AAGCAGTGGTATCAACGCAGAGTGCAGTGCTXXXXXXrGrGrG-3', where ‘‘rG’’ denotes a riboguanine and ‘‘XXXXXX’’ was a 6-bp barcode). Each well of the capture plate contained a different template-switching helper oligo with a distinct barcode. Plates were then frozen.
The cell capture plate was thawed, and 5 microlitre reverse transcription mix (4mM DTT, 2mM dNTP, 5U/microlitre SuperscriptII in 100 mM Tris-HCl at pH 8, 375 mM KCl, 0.1% Tween-20, 6 mM MnCl2, 2500 molecules of control mRNA) was added to each well. The synthetic mRNA consisted of 92 different ERCC control RNAs). The plate was incubated (10°C for 10 min, 42°C for 45 min) to complete reverse transcription and template switching. To purify the cDNA and remove unreacted primers, 100 microlitre MyOne carboxylatebeads (Invitrogen) was washed twice in 100 microlitre EBT (10 mM Tris-Cl at pH 8.5, 0.02% Tween-20) and then resuspended in 2 mL 14% PEG-6000 in 0.9 M NaCl, and 20 microlitre of this mixture was added to each well. Beads from all wells were pooled, washed in 70% ethanol twice, dried, and eluted in 37 microlitre EBT in a 1.5-mL polyallomer tube (Beckman). The cDNA was amplified in a single tube in 50 microlitre of 200 microM dNTP, 200 nM STRT-PCR primer (5’-biotin-AAGCAGTGGTATCAACGCAGAGT-3’), 1x Advantage2 DNA Polymerase Mix (Clontech) in 1x Advantage2 PCR buffer (Clontech) with 1 min at 95°C followed by 20 cycles of 15 sec at 95°C, 30 sec at 65°C, 4 min at 68°C,with heated lid.A 5 microlitre aliquot was amplified for another five cycles and visualized on a 1.2% agarose E-gel (Invitrogen) to confirm the range of cDNA lengths. The product was immobilized on MyOne C1 Streptavidin beads. Twenty microliters of beads was washed twice in 50 microlitre 2x BWT (10 mM Tris HCl at pH 7.5, 1 mM EDTA, 2 M NaCl, 0.02% Tween-20) and added to the remaining 45 microlitre PCR product. After a 10-min incubation at room temperature, the beads were washed three times in 50 microlitre 1x BWT and twice in EBT. Amplified cDNA was fragmented by DNase I in the presence of Mn2+. Beads were resuspended in DNase I buffer supplemented with 10 mM MnCl2 and DNaseI diluted to 0.0003U/microlitre in a total volume of 120 microlitre for exactly 8 min at room temperature. The reaction was stopped by washing the beads five times in 50 microlitre EBT. Beads were resuspended in 25 microlitre EBT, and 25 microlitre NEBNext End Repair reaction mix (New England Biolabs) was added; then the beads were incubated for 30 min at room temperature. The beads were washed twice in EBT, then resuspended in 21 microlitre EBT; 2.5 microlitre NEBNext dA tailing buffer and 1.5 microlitre Klenow exo- (both NEB) were added, and the reaction was incubated for 30 min at 37°C, followed by two washes in 50 microlitre EBT. An adapter containing the Illumina P2 sequence (5’-CAAGCAGAAGACGGCATACGAGCTCTTCCGATCT-3’ and 3’-PHO-GTTCGTCTTCTGCCGTATGCTCGAGAAGGCTAG-PHO-5’) was ligated by resuspending the beads in 25 microlitre EBT and adding 2x ligationmix (2x NEBuffer4, 2 microM adapter,1U/microlitre T4DNAligase,2U/microlitre SalI-HF, and 2mM ATP; all reagents from NEB) and incubating for 30min at 37°C and then washing twice in 25 microlitre EBT. The beads were then used as template in a 100 microlitre PCR reaction (200 microM dNTP, 400 nM each primer of primers 5’-AATGATACGGCGACCACCGAGATCTAAGCAGTGGTATCAACGCAGAGT-3’ and 5’-CAAGCAGAAGACGGCATACGAG-3’, 200 microM dNTP ,0.2U/microlitre Phusion polymerase in PhusionHF buffer ;all from NEB) with 30 sec at 98°C, followed by 12 cycles of [10 sec at 98°C, 30 sec at 65°C, 30 sec at 72°C] followed by 5 min at 72°C. The product was purified by AmPure XP (Beckman Coulter) and resuspended in 20 microlitre EBT. The sample was size-selected on a 2% E-gel with SYBR Safe (Invitrogen), recovering the 200 400 bp range by Qiaquick Gel Extraction (replacing the heating step with 15 min of vigorous agitation). The well indexing barcodes are read as bases 1-6 of the read, or for plates L281 and L282 as bases 5-10 of the read.
 
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina Genome Analyzer IIx
 
Description single cell
Data processing Read quality filtering and barcode extraction: Every read marked as valid by the Illumina control software was processed as follows: a) any 3' bases with a quality score of 'B' were removed; b) the 6 base well-identifying barcode was extracted from the 5' end of the read; c) the following bases should be template switch derived Gs. If the first three were not all Gs or two Gs and one uncalled base, the read was discarded. Else, these plus a maximum of six more consecutive Gs were removed; d) the remaining sequence should be transcript derived. If this ended in a poly(A)-sequence leaving less than 25 bases, the read was discarded; e) if the transcript-derived sequence consisted of less than six non-A bases, or a dinucleotide repeat with less than six other bases at either end, the read was discarded.
Alignment:the reads were aligned to the genome using the Bowtie1 aligner, allowing for up to three mismatches and up to 24 alternative mappings for each read. The genome included an artifical chromosome, containing a concatamer of the ERCC control sequences. Any reads with no alignments were re-aligned against another artifical chromosome, containing all possible splice junctions arising from the exons defined by the known transcript variants. Reads mapping within these splice junctions were translated back to the corresponding actual genomic positions. The UCSC transcript models were used for the expression level calculation. If a locus had several transcript variants, the exons of these were merged to a combined model that represented all expression from the locus. To account for incompete cap site knowledge, the 5' ends of all models were extended by 100 bases, but not beyond the 3' end of any upstream nearby exon of another gene of the same orientation.
Annotation and quantitation: The annotation step was performed barcode-by-barcode. Only reads mapping in sense orientation of an exon were considered as potentially transcript derived. Any such read that had one or more repeat mappings that was outside exons, was assigned randomly as one of these repeats and contributed to the summarized read count of that repeat class. Else, if it had one or more sense mappings to exons, it was assigned at the exon where it was closest to the transcript model 5' end, even if the sequence was repeat-like. If it had no exon mapping, it was assigned randomly at one of the mappings. The expression level of each transcript model was taken as the total number of reads in sense orientation at all its possible mapping positions.
Genome_build: UCSC mm9
Supplementary_files_format_and_content: Usoskin_DataTable.txt is a TAB-delimted text file containing Reads Per Million values for each analyzed feature in each well. Prefix 'r_' indicates repeat types and summarizes total expression from all repeat regions (from RepeatMasker) of that type. Suffix '_locX', X=1,2... indicates alternative loci expressing the same gene.
 
Submission date Jul 24, 2014
Last update date May 15, 2019
Contact name Sten Linnarsson
Organization name Karolinska Institutet
Department Medical Biochemistry and Biophysics
Lab Molecular Neurobiology
Street address Scheeles väg 1
City Stockholm
ZIP/Postal code 171 65
Country Sweden
 
Platform ID GPL11002
Series (1)
GSE59739 RNA-Seq of single cells from the mouse lumbar dorsal root ganglion
Relations
BioSample SAMN02942063
SRA SRX662072

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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