GSM6938338: BJ, WT, 9Months Cerebral organoid hBG_s50; Homo sapiens; RNA-Seq
Tissue samples were cryosectionned (20 µm sections collected at -20°C) and deposited on the double-barcoded DNA-arrays defined regions. During this process, slides were kept inside the cryostat chamber in order to conserve the integrity of the RNA and at -80°C for long storage.  Tissue sections were fixed with 4% Para-formaldheyde (PFA) (Life technologies 28908) in 1X PBS (Life technologies 70011-036) at room temperature during 10 minutes, then washed twice with 1X PBS, followed by a wash with double-distilled water (ddH2O). Tissues deposited on DNA arrays were covered with ddH2O and heat at 65°C during 5 minutes, followed by a fast chilling procedure (-20°C; 2 minutes). Remaining water was replaced by hybridization mix (10 U RNAse Inhibitor(New England Biolads M0314) in 6X SSC), and incubated at room temperature for 30 minutes. DNA arrays were washed by dipping them into a 50 mL falcon of 0.1X SSC then into a 50 mL falcon of ddH2O. Finally, tissue sections were air dried during 10 minutes. To avoid evaporation, reverse transcription (RT) is realised within sealed chambers (Thermoscientific small size; AB-0576). DNA arrays presenting tissue sections were incubated with RT mix (1 U/µL of SuperScript IV at (18090200 Life Technologies), 1X SSIV buffer (18090200 Life Technologies), 5 mM DTT (18090200 Life Technologies), 20 µg/mL actinomycin D (A1410-2MG Sigma), 0.2 mg/mL Bovine Serum Albumine (BSA) (Sigma Merk A9418-10G), a mix of dATP/dTTP/dGTP (130 µM each), 125 µM of dCTP and 5 µM dCTP-Cy3, 1 U/µL of RNAse Inhibitors) at 42°C overnight with a heated lid at 70°C. Next day, slides were washed by dipping them into a 50 mL falcon of 0.1X SSC, then into a 50 mL falcon of ddH2O and spin-dried. Finally, DNA arrays were scanned with the TRICT filter to reveal the presence of the fiducial borders as well as the physical position of tissue sections on top of the printed DNA arrays and revealed by the newly synthesized cDNA. After imaging, tissue sections were digested during 1 hour at 37°C and at 300rpm (20 mg/mL proteinase K (Invitrogen 4333793) at 7.5 mg/mL in PKD buffer (Quiagen 1034963)). Slides were washed in containers containing large volumes and under agitation (400 rpm) as following:  100 mL of preheated buffer 1 (2X SSC and 0.1X SDS (Sigma 71736); 50°C) during 10 minutes, 5 minutes with buffer 2 (0.2X SSC; room temperature), 5 minutres with buffer 3 (0.1X SSC; room temperature). Finally the slides were washed by dipping them in a 50 mL falcon containing ddH2O and spin-dried. DNA arrays were inspected at this stage to make sure that tissues was completely removed. DNA arrays were incubated with a freshly prepared 0.1 N NaOH solution during 10 minutes at room temperature, washed with the same solution, then neutralized for 2 minutes (for 100µL of 0.1 N NaOH, add 11.8 µL of 10X TE and, 6.5 µL of 1.25 M acetic acid). Finally, slides were washed by dipping them into a 50 mL falcon of 0.1X SSC, then into a 50 mL falcon of ddH2O and spin-dried. DNA arrays were incubated with a poly-C tailing mix ( 0.6 U/µL TDT (New England Biolads M0315), 1X terminal transferase buffer (New England Biolads BO315), 0.3X CoCl2 (New England Biolads B0252), 0.2 mM dCTP) during 35 minutes at 37°C, then 20 minutes at 70°C to deactivate the enzyme and cooled at 12°C. Under these conditions, the length of the poly-C tail should be of ~20 nts  in average (4). Then, slides were washed in a 50 mL falcon containing 0.1X SSC , then in a falcon containing ddH2O prior to be spin-dried.  To generate a complementary DNA strand, DNA arrays were incubated with a 2nd strand sythesis mix (0.1 U/µL  klenow exo- (Biolads M0212), 0.2 mg/mL BSA, 1X NEB2 buffer (New Englad Biolads B7002), 0.5 mM dNTPs) in presence of an oligonucleotide presenting a complementary sequence for the poly-C tailed sequence, as well as a 5'-extremity providing an adapter sequence (1µM conc: “GTTCAGACGTGTGCTCTTCCGATCTGGGGGGGGGH”). Second strand synthesis was performed within sealed chambers at 47°C during 5 minutes (primer anhealing), 37°C during 1 hour (extension),   10 minutes at 70°C (enzyme deactivation) and cooled et 12°C. Slides were washed in a 50 mL falcon containing 0.1X SSC and a second falcon containing ddH2O.  Newly synthesized complementary DNA strands were recovered by incubating DNA arrays with 100 µL of 0.1N NaOH solution. After 10 minutes of incubation, NaOH solution has been collected in a 1.5 mL eppendorf, DNA arrays were washed with other 100 µL of 0.1N NaOH solution and collected within the same eppendorf. The 200 µL collected NaOH solution has been neutralized with 23.6 µL of 10X TE and 13 µL of 1.25 mM acetic acid. DNA arrays were also neutralized with 100µL 0.1N NaOH solution mixed with 11.8µL 10X TE and 6.5µL 1.25mM acetic acid, then washed with 0.1X SCC and ddH2O for potential reuse if required.  Collected solution has been ethanol precipitated (2X EtOH volume,  200 mM NaCl, 1µL of 35 mg/mL glycogen; 1 hour at -20°C; centrifuged at 12000 rpm, washed with 70% EtOH) and resuspended in  23 µL of ddH2O for 15 minutes at room temperature.  The 23 µL of cDNA's complementary-probe strand solution in ddH2O has been mixed with 25 µL of Q5 hot start high fidelity 2X master mix(M0464L) , 1 µL adapter seq1 primer (GTTCAGACGTGTGCTCTTCCGATCT; 0.02 µM final conc.) and 1 µL adapter seq2 primer containing a part of the T7-promoter sequence (TACACTCTTTCCCTACACGACGCTCTTCCGATCTGACTCGTAATAC; 0.02 µM final conc.).   This mix has been PCR amplifyied (98°C, 30 sec; 15 cycles: 98°C 10 sec; 65°C 75 sec; then 65°C 5 minutes; 12°C hold), cleanned at 0.9X ratio with SPRIselect beads (Beckman Coulter B23318) and resuspended in 24µL of ddH2O.  A second round of PCR amplification has been performed by mixing the 24 µL of adaptors linked cDNA's complementary-probe strand with 25 µL 2X Q5 high fidelity ot start master mix, 0.5 µL of universal primer and 0.5 µL of index primer (each at 0.5µM concentration; NEBNext multiplex oligos for Illumina  index primers set 1 E7335). This mix has been amplified for 15 cycles (same amplification conditions as before), then cleanned at an 0.8X ratio with SPRISelect Beads (this ratio is used to get rid off fragments < 200nt because the DNA-array probes added to the illumina sequencing adaptors and barcodes is 223nt long without capturing any RNA). Final elution has been done in 25 µL of ddH2O.  5 µL of this library are used for quantitative PCR evaluation, targeting known transcript sequences (poly-A proximal regions), and the remaining 20 µL of this library is used for Illumina sequencing (150 nts paired-ends sequencing; NovaSeq). poly-T captured mRNA-seq performed with double-barcoded DNA arrays.