title: GSM8163334: sample 2 10X multiome (RNA) coronal section; Gallus gallus; RNA-Seq
library_construction_protocol: The telencephalon was dissected  from brains of 15 days old domestic chickens. Two different sample types of chicken brain were used to isolate nuclei: dissected telencephalon snap-frozen in liquid nitrogen and a cryosectioned sample (12x 50 µm sections). The following procedure was followed to extract the nuclei: the tissues were resuspended in 300 µl nuclei lysis buffer comprising 10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% Nonidet P40, 1 mM dithiothreitol and 1U/µl RNasin ribonuclease inhibitor (Promega) in nuclease-free water, pipeted until homogenized, 1 ml nuclei lysis buffer was added and samples were incubated on ice for 5 min. Nuclei were pelleted by centrifugation at 500 g for 5 min at 4°C and resuspended in 1x PBS, 1% BSA, 1U/µl RNasin ribonuclease inhibitor. Nuclei were centrifuged another time at 500 g for 5 min at 4°C and resuspended again in 1x PBS, 1% BSA, 1U/µl RNasin ribonuclease inhibitor. DAPI was added to the samples at a conc. of 0.5 µg/ml and incubated for 10 min. DAPI positive nuclei were sorted on a MA900 Sony cell sorter (Sony Biotechnology). The sorted nuclei were centrifuged at 500 g for 5 min at 4°C and resuspended in a 1 x nuclei buffer (10x Genomics) supplemented with 1 mM dithiothreitol and 1U/µl RNasin ribonuclease inhibitor. Single-cell libraries were generated using the 10X Chromium Single-Cell Instrument and NextGEM Single Cell Multiome ATAC+Gene Expression kit (10X Genomics) according to the manufacturer's protocol. In brief, the single nuclei of chicken brains were incubated for 60 min at 37°C with a transposase that fragments the DNA in open regions of the chromatin and adds adapter sequences to the ends of the DNA fragments. After generation of nanoliter-scale gel bead-in-emulsions (GEMs), GEMs were incubated in a C1000 Touch Thermal Cycler (Bio-Rad) under the following program: 37°C for 45 min, 25°C for 30 min, and hold at 4°C. Incubation of the GEMs produced 10x barcoded DNA from the transposed DNA (for ATAC) and 10x barcoded, full-length cDNA from poly-adenylated mRNA (for GEX). This was followed by a quenching step that stopped the reaction. After quenching, single-cell droplets were broken and the transposed DNA and full-length cDNA were isolated using Cleanup Mix containing Silane Dynabeads. To fill gaps and generate sufficient mass for library construction, the transposed DNA and cDNA were amplified via PCR: 72°C for 5 min; 98°C for 3 min; seven cycles of 98°C for 20 s, 63°C for 30 s, 72°C for 1 min; 72°C for 1 min; and hold at 4°C. The pre-amplified product was used as input for both ATAC library construction and cDNA amplification for gene expression library construction. Illumina P7 sequence and a sample index were added to the single-strand DNA during ATAC library construction via PCR: 98°C for 45 s; 7–9 cycles of 98°C for 20 s, 67°C for 30 s, 72°C for 20 s; 72°C for 1 min; and hold at 4°C. The sequencing-ready ATAC library was cleaned up with SPRIselect beads (Beckman Coulter). Barcoded, full-length pre-amplified cDNA was further amplified via PCR: 98°C for 3 min; 6–9 cycles of 98°C for 15 s, 63°C for 20 s, 72°C for 1 min; 72°C for 1 min; and hold at 4°C. Subsequently, the amplified cDNA was fragmented, end-repaired, A-tailed, and index adaptor ligated, with SPRIselect cleanup in between steps. The final gene expression library was amplified by PCR: 98°C for 45 s; 5–16 cycles of 98°C for 20 s, 54°C for 30 s, 72°C for 20 s, 72°C for 1 min; and hold at 4°C. The sequencing-ready GEX library was cleaned up with SPRIselect beads. Before sequencing, the fragment size of every library was analyzed using the Bioanalyzer high-sensitivity chip. All 10x scATAC libraries were sequenced on a NextSeq2000 instrument (Illumina) or on a NovaSeq6000 instrument (Illumina) with the following sequencing parameters: 51 bp read 1 – 8 bp index 1 – 24 bp index 2 – 51 bp read 2 and for 10x GEX libraries the following sequencing parameters were used: 28 bp read 1 - 10 bp index 1 - 10 bp index 2 - 90 bp read 2. OTHER: scRNA-seq + scATAC-seq