title: GSM8898657: WT; Danio rerio; RNA-Seq
library_name: GSM8898657
library_construction_protocol: Tissue sections were adhered to the Stereo-seq chip (generated by BGI, China) surface and incubated at 37℃ for 3 minutes. Then, the sections were fixed in methanol and incubated for 40 minutes at -20℃ before Stereo-seq library preparation. Where indicated, the same sections were stained with nucleic acid dye (Thermo fisher, Q10212) and imaging was performed with a Motic Custom PA53 FS6 microscope prior to in situ capture at the channel of FITC. After washed with 0.1x SSC buffer (Thermo, AM9770) supplemented with 0.05 U/ml RNase inhibitor (NEB, M0314L), tissue sections placed on the chip were permeabilized using 0.1% pepsin (Sigma, P7000) in 0.01 M HCl buffer, incubated at 37℃ for 5 minutes and then washed with 0.1x SSC buffer (Thermo, AM9770) supplemented with 0.05 U/ml RNase inhibitor (NEB, M0314L). RNA released from the permeabilized tissue and captured by the DNB was reverse transcribed overnight at 42℃ using SuperScript II (Invitrogen, 18064-014, 10 U/ml reverse transcriptase, 1 mM dNTPs, 1 M betaine solution PCR reagent, 7.5 mM MgCl2, 5 mM DTT, 2 U/ml RNase inhibitor, 2.5 mM Stereo-seq-TSO and 1x First-Strand buffer). After reverse transcription, tissue sections were washed twice with 0.1x SSC buffer and digested with Tissue Removal buffer (10 mM Tris-HCl, 25 mM EDTA, 100 mM NaCl, 0.5% SDS) at 55℃ for 10 minutes. cDNA-containing chips were then subjected to Prepare cDNA Release Mix (cDNA Release Enzyme, cDNA Release buffer) treatment for over night at 55℃. cDNA were purified using the VAHTSTM DNA Clean Beads (0.8×). The concentrations of the resulting PCR products were quantified by QubitTM dsDNA Assay Kit (Thermo, Q32854). A total of 20 ng of DNA were then fragmented with in-house Tn5 transposase at 55℃ for 10 minutes, after which the reactions were stopped by the addition of 0.02% SDS and gently mixing at 37℃ for 5 minutes after fragmentation. Fragmented products were amplified as described below: 25 ml of fragmentation product, 1x KAPA HiFi Hotstart Ready Mix and 0.3 mM Stereo-seq-Library-F primer, 0.3 mM Stereo-seq-Library-R primer in a total volume of 100 ml with the addition of nuclease-free H2O. The reaction was then run as: 1 cycle of 95℃ 5 minutes, 13 cycles of 98℃ 20 seconds, 58℃ 20 seconds and 72℃ 30 seconds, and 1 cycle of 72℃ 5 minutes. PCR products were purified using the AMPure XP Beads (0.63 and 0.153), used for DNB generation and finally sequenced on MGI DNBSEQ-Tx sequencer.
identifiers.namespace: GEO
identifiers.id: GSM8898657_r1