GSM8854805: GCs, Rbp4-Cre, SS2, DG_P28_E3_B6; Mus musculus; RNA-Seq
Brain slices from Rbp4-Cre:Ai9 mice were made using a 500 μm brain matrix, and the dentate gyrus (DG) was dissected. Next, the DG was chopped in smaller pieces and digested using Pronase. Following the digestion, the tissue pieces were homogenized using two-size Pasteur pipettes and the mix was loaded in a 20% Percoll solution to remove debris. Following centrifugation, the pellet was resuspended in OptiMEMtrehalose, and DAPI was added. Fluorescence Activated Cell Sorting (FACS) was done based on DAPI and tdTomato signal, into 96-well plates containing lysis buffer (see Methods for details). For the first strand synthesis, the Smart-Seq2 plates were thawed to room temperature for 1 min and subsequently spun down at 2000 xg for 1 min. The RNA denaturation was carried out at 72 °C for 10 min and flash-cooled on ice for 5 min. First strand synthesis mix (1X SuperScript II first strand synthesis buffer, Thermo Fisher; 5 mM DTT, Thermo Fisher; 100 U Oligo SuperScript II reverse transcriptase enzyme, Thermo Fisher; 10 U RNAse OUT, Thermo Fisher; 1M Betaine, Sigma Aldrich; 6 mM MgCl2, Thermo Fisher; and 1 μM TSO, IDT Technologies) is added to the cell lysis mix to a total volume of 10 μM. First strand synthesis reaction was carried out with following program: 42 °C for 90 min; 10 cycles of {50 °C for 2 min; 42 °C for 2 min}; 72 °C for 15 min; 4 °C indefinite hold. PCR amplification of the first strand product was performed by adding 15 µl of the PCR mix to the first strand product (1X KAPA HiFi HotStart Ready Mix, Roche; and 0.1 µM of IS PCR primer, IDT Technologies). The 22 cycles of the following PCR program was used to amplify the first strand product: 98 °C for 3 min; 22 cycles of {98 °C for 20 s; 67 °C for 15 s; 72 °C for 6 min}; 72 °C for 5 min; 4 °C indefinite hold. Using Hamilton STAR liquid handler (Hamilton), 20 µl of Ampure XP was added to each well and mixed. Standard Ampure XP purification was carried out as per the manufacturer's recommendation and the cDNA library was eluted in 22.5 µl of Elution buffer. 22 µl of the eluted library was transferred to fresh Echo Mass Spectrometry (MS) Qualified 384-well Polypropylene (PP) Microplate (Beckman Coulter). Labcyte Echo 550 acoustic dispenser (Beckman Coulter) was used for the next steps. 1 µl of Tn5 tagmentation mix (1X Tagment DNA buffer, Illumina; ATM mix, Illumina and cDNA library 1 ng) for cDNA fragmentation. Tn5 tagmentation was performed with the following program: 55 °C for 10 min; 4 °C indefinite hold. Tagmentation reaction was stopped by quenching the reaction with 500 nl of NT buffer for 5 min (Illumina). 750 nl of the i5 and i7 Illumina indexes were added to the plates. Finally, 2 µl of NPM master mix was added to the plate and mixed well and place for index PCR amplification: 72 °C for 3 min; 95 °C for 30 s; 12 cycles of {95 °C for 10 s; 55 °C for 30 s; 72 °C for 30 s}; 72 °C for 5 min, and 4 °C indefinite hold. The indexed PCR products are pooled in a single tube and 0.8X Ampure XP purification (Beckman Coulter) was carried out as per manufacturer's recommendation and finally the sequencing library was eluted in 35.5 µl of Elution buffer (Qiagen). Smart-Seq2 libraries were sequenced on NextSeq 500 (Illumina) sequencing platforms. Sequencing was done as per the protocol recommendations: paired end read of 61 bps (read 1), 61 bps (read 2), 8 bps (index 1) and 8 bps (index 2). Sequencing was performed at a depth of 500,000 reads per cell.