GSM8290047: SABERb2_brainD1, Medgenome_Run2, brain2, transcriptome; Danio rerio; RNA-Seq
Wild type and SABER x her4.3:switchCas9 zebrafish brains were harvested and dissociated at 21-23 dpf as described previously (Raj et. al 2018, Nature Biotechnology)  with the following modifications. All microcentrifuge tubes were treated with 1% BSA/DPBS overnight to prevent cell loss due to adhesion. Brains were dissociated with 1 mL of 20 units/ml papain in EBSS media and incubated at 37 °C for 20 min. Cells were resuspended with ~150 μl ice cold 1%BSA/DPBS solution. Transcriptome libraries were processed according to the manufacturer's instructions (Single Cell 3' v3 kit). Transcriptome libraries were sequenced using NovaSeq S4 300 cycle kits (Novogene) and Novaseq SP 100 and 200 cycle kits (MedGenome). To generate SABER-seq libraries, samples post cDNA amplification and prior to fragmentation were split into two parts. One part was processed for transcriptome libraries as instructed by the manufacturer. The other part was processed for SABER-seq libraries as follows. To enrich for SABER barcodes, 5 µl of the whole transcriptome cDNA was PCR amplified with 10X_p140v1UP  (GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT CGTCGTAGATCTTCATCGTGATG) and 10XPCR1F (CTACACGACGCTCTTCCGATCT) primers and Q5 polymerase. The reaction conditions were: 98 °C, 30 s; [98 °C, 10 s; 68 °C, 25 s; 72 °C, 15 s] x 14 cycles; 72 °C, 2 min. The reaction was cleaned up with 0.8X SPRISelect beads and eluted in 20 µl elution buffer (EB) buffer. A second PCR was carried out using 3 µl of PCR1 product and 10XPCR1F and 10X_p140v2Sa (GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT GAAACTACATGGGGTcaGCTC) primers. Reaction conditions were same as PCR1 except only 13 cycles were completed. At this point, 2 different clean up conditions were used: iteration 1 (SABER-seq_v0) and iteration 2 (SABER-seq_v1). For iteration 1, the reaction was cleaned up with 0.8X SPRISelect beads and eluted in 20 µl elution buffer (EB) buffer similar to PCR1.  For iteration 2, an optimization was trialed, and the reaction was cleaned up with right side size selection using SPRIselect beads (0.6X beads used initially and supernatant was rebound with 1.5X beads). The product was eluted in 20 µl EB. Finally, adapters and sample indices were incorporated in a third PCR reaction using 5-7 µl of a 1:10 dilution of PCR2 product. Reactions conditions were same as PCR1 except 9-10 cycles were completed. Primers used: 10XP5Dual_Hx (AATGATACGGCGACCACCGAGATCTACAC-xxxxxxx xxx-ACACTCTTTCCCTACACGACGCTCTTCCGATCT), 10XP7Dual_H5 (AATGATACGGCGAC CACCGAGATCTACAC-xxxxxxxxxx-ACACTCTTTCCCTACACGACGCTCTTCCGATCT), where x represents index bases from the 10X Dual Index Kit TT, SetA H index entries. For iteration 1, the third PCR reaction was cleaned up with 0.8X SPRISelect beads and eluted in 20 µl elution buffer (EB) to generate final libraries. For iteration 2, the third PCR reaction was cleaned up with right side size selection using SPRIselect beads (0.65X beads used initially and supernatant was rebound with 1.5X beads). SABER-seq libraries were sequenced using Novaseq SP 200 cycle kits (MedGenome) with 5-10% PhiX spike-in. Sequencing parameters: Read1 28 cycles, Read2 151 cycles, Index1 10 cycles, Index2 10 cycles. Standard sequencing primers were used.