GSM1444961: L130_F05; Mus musculus; RNA-Seq
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.
GEO Accession
GSM1444961